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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1481-1487.)
© 1996 American Heart Association, Inc.

Articles

LDL Stimulates Chemotaxis of Human Monocytes Through a Cyclooxygenase-Dependent Pathway

Jorg Kreuzer; Stefanie Denger; Lothar Jahn; Jorg Bader; Kai Ritter; Eberhard von Hodenberg; Wolfgang Kubler

the Innere Medizin III, Universitat Heidelberg, Heidelberg, Germany.

Correspondence to Jorg Kreuzer, MD, Universitat Heidelberg, Innere Medizin III, Bergheimer Str 58, 69115 Heidelberg, Germany.

	Abstract

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Monocyte migration into the vessel wall is an early step in atherogenesis. Even though a number of chemotactic factors have been identified, the regulation of the chemotactic response is not clearly understood. As the release of arachidonic acid has been implicated in monocyte chemotaxis, we studied the influence of LDL, which can supply this fatty acid to cells, on the chemotactic mobility of monocytes. Migration of human monocytic U937 cells was abolished by a 30-hour incubation in medium containing lipoprotein-depleted 10% fetal calf serum. Thereafter, human VLDL, LDL, acetyl LDL, methyl LDL, HDL, free cholesterol, linoleic acid, oleic acid, or arachidonic acid was added. At the end of varying incubation periods (0.5 to 8 hours), chemotaxis, viability, and cellular cholesterol content were measured. In the same experimental setting we also studied the effects of the pharmacological agents chloroquine, indomethacin, and acetylsalicylic acid on LDL-mediated chemotaxis. Chemotaxis was restored by LDL in a dose- and time-dependent manner starting at concentrations as low as 5 µg/mL and at incubations as brief as 30 minutes. The other lipoproteins tested (VLDL, HDL, acetyl LDL, and methyl LDL) as well as free cholesterol had no comparable effect on chemotaxis. Viability and total cholesterol content did not differ among the groups. Simultaneous incubation of cells with chloroquine, indomethacin, and acetylsalicylic acid reduced restitution of chemotaxis by LDL by 71%, 82%, and 68%, respectively. In contrast, the agents had only slight inhibitory effects on the chemotactic mobility of serum-fed control cells. Incubation with linoleic acid showed a 60% restoration of chemotaxis, whereas arachidonic acid stimulated chemotaxis by 140% compared with the positive control. Preincubation of LDL with the monoclonal antibody MB47 directed against LDL resulted in a significantly reduced migratory response. The data suggest a novel cyclooxygenase-dependent regulatory mechanism of chemotaxis by LDL.

Key Words: chemotaxis • U937 cells • LDL • arachidonic acid • cyclooxygenase

	Introduction

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Monocyte chemotaxis is a highly regulated process that results in immigration of mononuclear cells at sites of immune response.¹ For the development of atherosclerosis, migration of monocytes into the vessel wall also appears to play a pivotal role. Monocytes have been demonstrated in very early atherosclerotic lesions.²

Migration of monocytes and hence their chemotactic response depend on two mechanisms. The first is the presence or absence of specific chemoattractants, such as monocyte chemotactic protein-1³ ; the second is the susceptibility of cells to migrate toward a chemotactic gradient. This study investigated factors that may influence the chemotactic susceptibility of monocytes.

A number of mechanisms influencing the chemotactic ability of monocytes have been studied. Induction of certain receptors contributes to the ability of the cells to migrate,⁴ and the number of receptors for a certain chemotactic stimulus is directly related to the chemotactic response. Apart from ligand-receptor interaction, which can regulate cell motion, the fluidity of the cell membrane, depending on its cholesterol content, can mediate chemotactic susceptibility.⁵ This may, at least in part, be attributed to an influence of cholesterol on the function of certain proteins in the cell membrane.^{6 7} The physiological implications of this observation, however, remain elusive since plasma membrane cholesterol content does not appear to vary extensively in vivo. The susceptibility of different cells to chemotaxis is dependent on metabolites from the mevalonate pathway.^{8 9} Locati et al¹⁰ describe a rapid release of AA in monocytes by monocyte chemotactic protein-1. When AA release from phospholipids was blocked by phospholipase A2 inhibitors, the chemotactic response of monocytes was abolished, indicating that this release is a prerequisite for chemotaxis of monocytes.

As the major carrier of cholesterol in humans,¹¹ LDL plays a key role in atherogenesis (see Reference 2 for review). Data from this laboratory have provided the first evidence that LDL can in fact regulate chemotaxis of mononuclear cells.⁸ The mechanism, however, remains elusive. Other authors have shown that LDL, besides providing cholesterol and phospholipids to cells, can also function as a donor of AA for eicosanoid synthesis.^{12 13} It was therefore the aim of this study to investigate whether LDL-induced chemotaxis is linked to AA supply and prostanoid synthesis and whether LDL, by providing AA, could stimulate cellular migration.

We now report that LDL stimulates the chemotactic susceptibility of monocytic cells in a dose- and time-dependent manner. All LDL effects on monocyte chemotaxis could likewise be induced by AA. The experiments further suggested that induction of chemotaxis by LDL depends on prostanoid synthesis through cyclooxygenase rather than on the presence of free AA.

	Methods

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Lipoprotein Isolation
VLDL (d=1.006 to 1.019 g/mL), LDL (d=1.019 to 1.063 g/mL), and HDL (d=1.073 to 1.21 g/mL) were prepared from the sera of normal fasted subjects,¹⁴ dialyzed extensively against 0.01 mol/L sodium phosphate, pH 7.4, containing 0.15 mol/L sodium chloride and 0.01% EDTA, filter-sterilized, and stored at 4°C. Storage and dialysis were performed under a nitrogen atmosphere to avoid oxidation. All procedures were performed under sterile, pyrogene-free conditions to prevent lipopolysaccharide contamination. The lipopolysaccharide content of representatively prepared samples was continuously monitored.¹⁵ All lipoproteins were used within 10 days after preparation to prevent spontaneous modification of the lipoproteins.^{16 17} LPDS was prepared from heat-inactivated FCS by repeated centrifugation at d=1.21 g/mL at 4°C. After all lipoproteins were removed, the serum was dialyzed and filtered as described above, and aliquots were stored at -20°C. Protein concentrations were determined by using the method of Lowry et al¹⁸ ; cholesterol content was assessed by using a Boehringer Mannheim Monotest kit.

Methylation¹⁹ and acetylation²⁰ of lipoproteins were performed as described, and lipoproteins were analyzed for modification by using agarose gel electrophoresis.

Subjects
Normal subjects at the University of Heidelberg were recruited from the staff and student body as donors for lipoproteins. No subjects received drugs that might have affected lipid metabolism, and all had normal serum cholesterol and triglyceride levels. Informed consent was obtained from each subject.

Cell Culture
U937 cells, received from Dr A. Habenicht, University of Heidelberg, were cultivated at 37°C with 5% CO₂ in RPMI 1640 with L-glutamine 2 mmol/L plus 10% heat-inactivated FCS, penicillin 100 U/mL, and streptomycin 100 µg/mL, hereafter designated medium 2. RPMI 1640 without any additives was designated medium 1. Cell density was kept at 1 to 8x10⁵/mL, and fresh medium was added every 48 hours. Cell number was determined by using a computer-assisted flow cytometer (Scharfe), and the percentage of viable cells was assessed by using trypan blue dye exclusion.

Chemotaxis Assay
After the cells were washed three times in medium 1 to remove FCS, they were placed in RPMI 1640 with L-glutamine containing 10% LPDS (designated medium 3) at a density of 2x10⁵ cells/mL. The cells were incubated for 30 hours, and lipoproteins, free cholesterol, or fatty acids were added at the concentrations and for the times indicated in "Results." Cholesterol was taken from a stock solution prepared at 10 mg/mL in ethanol. Fatty acids were obtained from Nucheckprep (Elysian), and stock solutions of 10 mmol/L were prepared in ethanol. The purity of all fatty acids used was >99.9%. Chloroquine, indomethacin, or ASA was added to cells after 28 hours in medium 3, thus preincubating the cells with the compounds 2 hours before the addition of lipoproteins. Indomethacin was prepared fresh before each experiment at a concentration of 1 mg/mL in Tris buffer (0.1 mol/L, pH 8.6) and was used at 20 µg/mL; the final concentrations of ASA and chloroquine were 100 and 60 µmol/L, respectively. At the end of each incubation, the cells were washed three times in medium 1, and cell density was adjusted to 1x10⁶ cells/mL in the same medium.

In a second approach the uptake of LDL, instead of the degradation, was inhibited. The mAb MB47 (kindly provided by Linda Curtiss, The Scripps Research Institute, La Jolla, Calif) directed against LDL was preincubated with LDL for 2 hours at 37°C at a concentration of 20 µg/mL. As a control, an anti-transferrin mAb (20 µg/mL) was used. Fractions of cells were analyzed for migratory response and cholesterol content. Chemotaxis of cells was assessed by using a 48-well multiwell chamber (Nucleopore)²¹ with 1% human serum as chemoattractant.⁸ The pore size of the membrane used in all experimental settings was 5 µm. The chemotaxis chamber was incubated for 90 minutes in a humidified cell incubator. Migrated cells were stained by using the Hemacolor Set (Merck) and counted at a magnification of x320. A minimum of three wells per experiment were used; seven high-power fields/well were counted. Checkerboard assays were performed to distinguish between directed migratory response (chemotaxis) and random cell movement (chemokinesis). All experiments were performed in triplicate or quadruplicate. Results are given as the average number of migrated cells in percent of migratory response of control cells cultivated in medium 2.

Lipid Extraction and Analysis
Lipid extraction was performed according to a modification of the procedure of Folch et al.²² Analysis was performed by using TLC.²³ Defined volumes of cell homogenates were extracted, and samples were applied to high-performance TLC plates (10x20 cm silica gel; 60F 254/Merck). The spots were detected by using manganese chloride–sulfuric acid and quantified by scanning the high-performance TLC plates under fluorescent light (excitation at 366 nm, emission maxima at 410 nm) with the CAMAG TLC scanner II (CAMAG).

All materials were obtained from Sigma (chemicals) or GIBCO for tissue culture unless otherwise indicated. Data are presented as mean±SEM.

Gas Chromatography
For determination of cellular AA content, a modified protocol of McDonald-Gibson was used.²⁴ Briefly, fatty acids were extracted by using 1 mg/mL butylated hydroxytoluene (Sigma) in ethanol, 500 µg/mL pentadecanoic acid (Sigma) in n-heptane, a 2:1 (vol/vol) chloroform/methanol mixture, and 0.02% CaCl₂ two times and dried under nitrogen. Transmethylation was performed with 0.5 mol/L methanolic NaOH and 14% bortrifluoride-methanol complex (Merck). After drying under nitrogen the samples were stored at -20°C until used for gas chromatography. The AA content of the cells was analyzed by using an HP 5890 (Hewlett Packard) with a DB-223 fused-capillary column (30x0.25, 0.25 µm) (J&W) and quantified by using HP 3365 chemstation software. Cellular AA was calculated in nanograms per microgram cell protein.

	Results

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Migration Arrest
For the experiments the human monocytic cell line U937, a well-established model to study chemotaxis,²⁵ and LDL receptor–mediated lipoprotein uptake²⁶ were used. As it was necessary to also detect slight changes in migratory response, migration of cells was arrested in LPDS before the addition of LDL. Cell proliferation and viability were not significantly different for cells cultivated in medium 2 or 3 (as shown previously⁸ ). To define the time necessary to abolish chemotaxis, cells were incubated in medium 3 for varying times. After 30 hours of incubation no spontaneous chemotactic response of the cells was observed (6±4% chemotactic susceptibility in LPDS compared with 110±12% in FCS). Trypan blue dye exclusion demonstrated similar viability of cells grown in medium 2 or 3 for 30 hours (data not shown). According to these results all further experiments were performed after preincubation for 30 hours. Checkerboard assays revealed that the basic migratory response as well as the LDL and AA response observed was indeed chemotactic rather than chemokinetic (Table 1).

View this table: [in this window] [in a new window]   Table 1. Checkerboard Assay Testing for Chemotaxis and Chemokinesis of U937 Cells

Time Course
To investigate the time course of restoration of chemotaxis, LDL was added at a final concentration of 20 µg/mL (cholesterol content) for periods from 30 minutes to 4 hours. Chemotaxis began to recover 30 minutes after the addition of 20 µg/mL LDL (Fig 1a). After 4 hours of incubation, chemotactic response reached the level observed before growth arrest. Incubation beyond 4 hours had no additional effect on chemotaxis. When free cholesterol was used in the same experiments only a slight stimulating effect on chemotaxis was observed.

View larger version (30K): [in this window] [in a new window]   Figure 1. Line graphs. After migration arrest in RPMI 1640 medium with 10% LPDS, U937 cells were incubated with (a) LDL or free cholesterol (20 µg/mL each) for varying times or (b) varying concentrations of LDL or free cholesterol for 2 hours. After incubation the cells were washed in PBS, and chemotaxis to 1% human serum was assessed by using a 48-well microchemotaxis chamber. Values are mean±SEM (n=4) and are given as percent of migration of control cells grown in medium containing 10% FCS.

Dose Response of LDL and Effects of Other Lipoproteins
Chemotaxis could be stimulated when different concentrations of LDL (as low as 2.5 to 5 µg/mL) were incubated with U937 cells. Maximal chemotaxis was observed at LDL cholesterol concentrations of 20 µg/mL; free cholesterol added at identical concentrations revealed no comparable effect (Fig 1b). To define the specificity of the observed effect, lipoproteins other than LDL were also used for the incubation. VLDL, which can also be taken up by the scavenger receptor,¹¹ or HDL applied in identical concentrations as LDL induced only 20% or 40%, respectively, of the chemotactic response compared with the effect of LDL (Fig 2). To investigate whether the observed effect was dependent not only on the lipoprotein particle but also on its uptake by the LDL receptor, experiments using modified LDL were performed. Acetylated and methylated LDL were used to study the effects of unspecific uptake of LDL particles by the cells. Neither of these modified lipoproteins showed any significant effect on cell migration (Fig 2).

View larger version (44K): [in this window] [in a new window]   Figure 2. Bar graph. LDL but not VLDL, HDL, acetyl LDL, or methyl LDL led to complete restoration of chemotaxis. U937 cells were migration arrested in RPMI medium with 10% LPDS, and lipoproteins (20 µg/mL) were added for 2 hours. Before the migration assay cells were washed with PBS, and chemotaxis to 1% human serum was assessed. Values are mean±SEM (n=4) and are given as percent of migration of control cells grown in medium containing 10% FCS.

Fatty Acids
The fatty acid AA, a component of LDL, was added to the cells for 2 hours before chemotaxis was assayed. By using this approach it was possible to investigate whether chemotaxis could be affected by AA or substrates originating from AA. In fact, incubation with AA resulted in a highly significant stimulation of chemotaxis (140% of control) (Fig 3). Linoleic acid and oleic acid, which were used as controls because they are not a substrate for cyclooxygenase, did not exhibit a similar effect on the chemotaxis of U937 cells (Fig 3).

View larger version (44K): [in this window] [in a new window]   Figure 3. Bar graph. AA, linoleic acid, or oleic acid (10 µmol/L each) was added to cells for 2 hours after they were migration arrested for 30 hours in RPMI medium with 10% LPDS. Before the migration assay cells were washed with PBS, and chemotaxis to 1% human serum was assessed. For experiments with indomethacin (20 µg/mL) the compound was added 2 hours before AA or linoleic acid. Chemotaxis is expressed as percent of migration of control cells cultivated in medium with 10% FCS. Values are mean±SEM (n=3).

Chloroquine, Indomethacin, and ASA
To further characterize the connection between AA metabolism and LDL-induced migration, endosomal degradation of LDL was inhibited by using chloroquine. The inability of the cells to hydrolyze LDL suppressed stimulation of chemotaxis by >70% (Fig 4). In subsequent experiments prostanoid synthesis was blocked by adding indomethacin or ASA to the cultures. Again, only a minor migratory response was observed. Indomethacin blocked 80% and ASA 70% of LDL-induced chemotaxis. All compounds used had only slight effects on chemotaxis of U937 control cells that were cultured in medium 2 (Fig 4). In contrast, preincubation of LDL with an anti-LDL mAb resulted in significantly reduced chemotactic response of U937 cells, whereas the anti-transferrin mAb that was used as a control had no influence on migration (Fig 5).

View larger version (45K): [in this window] [in a new window]   Figure 4. Bar graphs. Restoration of chemotaxis by LDL after cultivation of cells in medium with 10% FCS or 10% LPDS was abolished through inhibition of lysosomal degradation of LDL and inhibition of cyclooxygenase. After incubation for 28 hours in either FCS or LPDS medium as described in "Methods," chloroquine (60 µmol/L), indomethacin (20 µg/mL), or ASA (100 µmol/L) was added for 2 hours. Either LDL (20 µg/mL; cells in LPDS medium) or buffer alone (cells in FCS medium) was added for an additional 2 hours, and chemotaxis was assessed. Values are mean±SEM (n=3) and are given as percent of migration of control cells grown in medium containing 10% FCS.

View larger version (59K): [in this window] [in a new window]   Figure 5. Bar graph shows inhibition of restoration of LDL-mediated chemotaxis by using the anti-LDL mAb MB47. Before being added to migration-arrested U937 cells, LDL (20 µg/mL) was incubated with MB47 for 2 hours. An anti-transferrin mAb (AT MAB) was used as a control. Cells were then incubated with LDL for 2 hours, and chemotaxis was assessed as described in "Methods." Values are mean±SEM (n=3) and are given as percent of migration of control cells grown in medium containing 10% FCS.

Cholesterol Content
After cells were incubated in LPDS medium for 30 hours, a significant decrease in cellular cholesterol was observed. Analysis of cellular cholesterol content showed free but not esterified cholesterol; these data are consistent with previous results.^{7 27} After addition of LDL or free cholesterol, a similar but only moderate rise in cellular cholesterol was observed (Table 2). Incubation with acetyl and methyl LDL led to modest increases in cellular cholesterol content, most likely through unspecific, non–receptor-mediated uptake of lipoproteins, as both ligands are not recognized by the LDL receptor, and undifferentiated U937 cells do not express the scavenger receptor that can mediate uptake of acetyl LDL. This is consistent with the results obtained by others who have shown that methyl LDL²⁸ and acetyl LDL²⁶ are not taken up by U937 cells through a receptor-dependent pathway.

View this table: [in this window] [in a new window]   Table 2. Total Cholesterol of U937 Cells After Incubation in FCS and LPDS Before and After Addition of Free Cholesterol and Lipoproteins

AA Content
To study the effect of LDL and arachidonate on cellular AA content, gas chromatography was used to measure cellular AA content. It could be shown that incubation of U937 cells with AA resulted in a significant rise in cellular AA of almost 100%. Incubation with LDL did not lead to a measurable increase in cellular AA (Table 3).

View this table: [in this window] [in a new window]   Table 3. AA Content of U937 Cells Under Different Culture Conditions

	Discussion

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Recent work suggests a crucial role of AA release from phospholipids for chemotactic response of monocytes.^{10 29} The free form of AA can either be reesterified into lipids or metabolized into leukotrienes and prostaglandins by lipoxygenases or cyclooxygenases. There is evidence that LDL, besides providing cholesterol, may also supply AA to the cyclooxygenase pathway for eicosanoid synthesis.^{12 13} We therefore investigated whether uptake of LDL via the LDL receptor pathway could render cells that are prone to migrate toward a chemotactic stimulus. Studies were performed to determine whether chemotactic response was related to AA supply. The present data indicate that the classic LDL pathway of Brown and Goldstein³⁰ plays a regulatory role in the chemotactic response of monocytic cells.

Previous studies suggest a major role of cholesterol in cellular chemotaxis. It has been hypothesized that migratory susceptibility is dependent on the fluidity of cell membranes. When cholesterol synthesis is blocked by using triparanol, chemotaxis can be arrested.⁵ In the present study incubation of cells in LPDS medium containing only low amounts of cholesterol (9 mg/mL) resulted in a reduction of cellular cholesterol content, as described previously,⁷ and an almost complete loss of chemotaxis. De Pace and Esfahani³¹ have shown that depletion of cellular cholesterol leads to changes in cell membrane function and a loss of microvilli. The migratory arrest in our experiments could not be attributed to cholesterol loss alone, however, as chemotaxis could be restored by LDL independent of cellular cholesterol content. Thus it was the aim of this study to investigate the effects of fatty acids on chemotaxis independent of the cholesterol content of the cells. Hence it appeared mandatory to assess the cholesterol supply through LDL to account for possible effects of sterols on chemotaxis. In contrast to chemotactic susceptibility, cellular cholesterol content was similar in cells incubated with LDL, acetyl LDL, methyl LDL, and free cholesterol. Although chemotaxis was stimulated after only 30 minutes of LDL incubation (Fig 1a), no significant effect on cellular cholesterol was observed (Table 2). These data provide strong evidence that cholesterol did not induce stimulation of chemotaxis.

The results obtained with modified forms of LDL strongly suggest that not only the lipoprotein but also the pathway of uptake is important for the observed restoration of chemotaxis. In human monocytes different lipoproteins, including LDL, are directed to different intracellular localizations after receptor-mediated uptake.³² Therefore it can be speculated that LDL receptor–mediated uptake of LDL directs contents of the LDL particle to certain cellular pools, suggesting that not only the amount but the localization of the internalized lipoprotein particle could be important for the observed effects.

This hypothesis is further supported by experiments using chloroquine, which inhibits lysosomal degradation of LDL. Chloroquine abolished LDL-mediated chemotaxis but had no effect on chemotaxis of control cells. Likewise, indomethacin and ASA, inhibitors of cyclooxygenase, exclusively inhibited LDL-induced chemotaxis. In addition, cell migration could be abolished by preincubation of LDL with the mAb MB47, whereas an anti-transferrin mAb used as a control had no comparable effects. When Kelley et al³³ studied the influence of different lipoprotein classes on activation of human monocytes using glucuronidase activity as an indicator of activation, they found the highest activity was induced by LDL; they also demonstrated a stimulation of prostaglandin E secretion by this lipoprotein. As the observed effects were inhibited by indomethacin they hypothesized that AA metabolism plays an important role in this process. According to these data, we could demonstrate that LDL, but not VLDL, HDL, or free cholesterol, despite leading to comparable effects on cellular cholesterol, induced complete restitution of chemotaxis. The three fatty acids used in this study displayed significantly different effects on chemotaxis. Whereas AA led to a strong restoration of chemotaxis that was even higher than the LDL-induced response, linoleic acid and oleic acid did not exhibit a comparable induction.

A role of free AA for cellular chemotaxis response has been suggested by Locati et al.¹⁰ They and others³⁴ have hypothesized that AA, rather than being a substrate for cyclooxygenase, could directly interact with cellular signals inducing migratory response, adhesion, or activation of the cells. The results of the present study clearly indicate that AA can also cause a rapid increase of chemotactic susceptibility through stimulation of the cyclooxygenase pathway. LDL, but not modified LDL, which is not taken up by the LDL receptor, provides the substrate for this stimulating effect. Very low amounts of LDL-supplied AA appear to be sufficient for this effect. For this reason it was not surprising that, despite a visible effect on prostaglandin metabolism, we detected no measurable changes in cellular arachidonate content with gas chromatography. Other authors, who incubated cells with much higher amounts of LDL for periods up to 24 hours, have been unable to measure a cellular rise in AA³⁵ ; however, this does not imply that arachidonate cannot be provided by LDL. By using radiolabeled arachidonate incorporated into reconstituted LDL particles, Habenicht et al¹² and Salbach et al¹³ have clearly demonstrated arachidonate supply to cells by LDL. Their observations, together with our data using inhibitors of prostaglandin synthesis and free AA, strongly suggest a major role of arachidonate in the regulation of chemotactic susceptibility.

As U937 cells cannot synthesize leukotrienes,^{36 37} it can be ruled out that the observed regulation of chemotaxis by LDL is connected to leukotriene synthesis, but rather depends on prostanoid synthesis.

Our results agree with other investigators who have studied the effect of phospholipase 2 inhibition. Phospholipase 2 mobilizes AA from the membrane and thus promotes prostanoid synthesis. If this enzyme is inhibited, mitigation of chemotactic response can be observed.^{10 38} In our experiments all LDL effects observed could be reproduced by adding AA but not linoleic acid or oleic acid to the cells. A number of authors have demonstrated that undifferentiated U937 cells, like the ones used in our experiments, cannot mobilize substantial amounts of AA from their membranes, thus making them dependent on an exogenous supply of this fatty acid.^{37 39 40} Hence, U937 cells are highly dependent on exogenous AA, as they are able to synthesize significant amounts of prostaglandins only when provided with exogenous AA.⁴⁰ This may explain why small amounts of exogenous arachidonate supplied by LDL led to a significant response of the cells. Thus, the results of this study strongly support the notion that LDL exhibits its effect on chemotaxis through a supply of AA.

Taken together, the current findings demonstrate a specific cholesterol-independent stimulation of monocytic chemotaxis by LDL. This effect depends on LDL uptake by the LDL receptor and on cyclooxygenase activity. The data suggest a direct implication of cyclooxygenase for this mechanism and support a new role of LDL in the regulation of chemotaxis of monocytic cells. This mechanism may be important for the understanding of the physiological role of LDL as well as for its role in the process of atherosclerosis.

	Selected Abbreviations and Acronyms

 

AA = arachidonic acid ASA = acetylsalicylic acid FCS = fetal calf serum LPDS = lipoprotein-deficient serum mAb = monoclonal antibody PBS = phosphate-buffered saline TLC = thin-layer chromatography

	Acknowledgments

 
This work was supported in part by SFB grant 320 and the Sandoz Stiftung. We thank Dr A. Habenicht for his gift of U937 cells and Marion Forster and Karin Bucher for excellent technical assistance. We are also in debt to Linda Curtiss, The Scripps Research Institute, La Jolla, Calif, for providing the MB47 antibody and Alex Dugrillon, Department of Clinical Chemistry, University of Heidelberg, Germany, for the quantification of AA.

Received February 28, 1996; revision received April 12, 1996;

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