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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:47-53

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:47-53.)
© 1999 American Heart Association, Inc.

Original Contributions

Tissue Factor Expression of Human Monocytes Is Suppressed by Lysophosphatidylcholine

Bernd Engelmann; Susanne Zieseniss; Korbinian Brand; Sharon Page; Arnd Lentschat; Artur J. Ulmer; Eckehart Gerlach

From the Physiologisches Institut der Universität München (B.E., S.Z., E.G.), Munich; Institut für Klinische Chemie und Pathobiochemie der TU München (K.B., S.P.), Munich; and Zelluläre Immunologie, Forschungszentrum Borstel (A.L., A.J.U.), Germany.

Correspondence to Dr Bernd Engelmann, Physiologisches Institut der Universität München, Pettenkoferstrasse 12, D-80336 München, Germany. E-mail Bernd.Engelmann{at}med.uni-muenchen.de


*    Abstract
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 *Abstract
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Abstract—The expression of tissue factor (TF), the principal initiator of coagulation, is increased during inflammation and atherosclerosis. Both conditions are promoted by lysophosphatidylcholine (lysoPC). We observed in the present study that lysoPC (1 to 10 µmol/L) dose-dependently reduced TF activity in human monocytes, as elicited by lipopolysaccharide (LPS). Lysophosphatidylethanolamine (lysoPE) and other lysophospholipids did not affect LPS-induced TF activity of human monocytes. TF antigen expression as elicited by LPS was also lowered by lysoPC. Phospholipid analyses indicated a selective increase in the lysoPC content of the monocytes after preincubation with the lysophospholipid. LysoPC inhibited the TF activity of Mono Mac-6 cells to a similar extent as in the monocytes. LPS binding to plasma membrane receptors and internalization of LPS into monocytes were not affected by lysoPC. In contrast, LPS-mediated nuclear binding of nuclear factor-{kappa}B/Rel to a TF-specific {kappa}B site was inhibited by lysoPC. Induction of TF mRNA expression by LPS tended to be partially reduced by the lysophospholipid. Preincubation with lysoPC increased monocytic cAMP levels. Inhibition of adenylyl cyclase by pretreatment with 2'-deoxy-3'-adenosine monophosphate partially reversed the inhibition of TF activity promoted by lysoPC. In conclusion, lysoPC markedly decreases LPS-mediated TF expression of human monocytes, the effect probably being mediated by both transcriptional and posttranscriptional mechanisms. LysoPC may thus attenuate activation of coagulation during inflammation and atherosclerosis.


Key Words: atherosclerosis • inflammation • lipopolysaccharide • cAMP • nuclear factor-{kappa}B


*    Introduction
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 up arrowAbstract
 *Introduction
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Tissue factor (TF) is the primary activator of the extrinsic and intrinsic pathways of coagulation (for recent reviews, see References 1 and 21 2 ). Within the extrinsic pathway of coagulation, TF forms a complex with factor VIIa that stimulates the generation of factor Xa, which in turn is necessary for the formation of thrombin from prothrombin. Additionally, the TF–factor VIIa complex activates the intrinsic coagulation pathway by stimulating the formation of factor IXa. Increased expression of TF is a feature of several pathophysiological conditions, in particular, inflammation and atherosclerosis. Agents known to induce or sustain inflammation, such as bacterial lipopolysaccharide (LPS), interleukin-1, tumor necrosis factor-{alpha} (TNF-{alpha}), or C-reactive protein, are among the most potent stimulators of TF expression.2 The increased activity of endothelial TF is thought to be responsible for the development of disseminated intravascular coagulation during sepsis.3 4 Furthermore, it has been proposed that TF contributes to the thrombotic complications associated with atherosclerosis. In agreement with this hypothesis, increased expression of TF has been observed in specimens taken from atherosclerotic plaques.5 6 In particular, macrophage TF has been implicated as the principal determinant for the thrombogenicity of unstable atherosclerotic plaques.7 8 9 Oxidized LDL, which is thought to be a causative factor in the pathogenesis of atherosclerosis, was shown to augment basal and LPS-induced TF activity of endothelial cells and monocytes, respectively.10 11

Local and systemic inflammatory processes are initiated and/or promoted by activation of phospholipase A2 (PLA2), in particular, those of the group II secretory type.12 PLA2-catalyzed hydrolysis of phosphatidylcholine (PC), the major phospholipid of most cells, liberates arachidonic acid, which may be further processed to eicosanoids. The second product of the reaction is lysophosphatidylcholine (lysoPC). This lysophospholipid is presumed to play a role in sustaining inflammation due to transcriptional activation of genes coding for adhesion molecules, cytokines, and growth factors.13 14 LysoPC is also formed in considerable amounts by oxidation of LDL particles, probably by PLA2-dependent hydrolysis of LDL-associated PC.15 Several of the atherogenic effects of oxidized LDL, eg, impairment of endothelium-dependent vasorelaxation16 or enhanced adhesion of monocytes to the endothelium,13 have previously been shown to be mediated by lysoPC. Thus, lysoPC is most probably generated in substantial amounts under pathophysiological conditions, when increased expression of TF is observed. In the present study we therefore wished to ascertain whether lysoPC affected monocytic TF expression.


*    Methods
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 *Methods
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Isolation of Monocytes
Human peripheral blood mononuclear cells (PBMCs) were isolated by buoyant density centrifugation with Ficoll-Hypaque essentially as described in Reference 1717 . Buffy coats obtained from healthy donors (anticoagulated by EDTA) were diluted with 3 vol of calcium-free PBS, and the suspension was underlayered with low-endotoxin Ficoll (d=1.077 g/mL, Pharmacia Biotech). After centrifugation for 25 minutes at 420g, the interphase was collected and washed twice with isotonic PBS containing 0.13% EDTA and 0.15% BSA (pH 7.4), which is referred to throughout this article as "washing buffer." The cell pellet obtained after the last centrifugation was usually suspended in 10 mL plasma concomitantly obtained from the same donor. To this suspension, first 50 µL of 9% NaCl was added, after 10 minutes an additional 100 µL was added, and after a further 10 minutes another 100 µL of 9% NaCl was added. The cells suspended in the hypertonic medium were overlayered on Ficoll (d=1.077 g/mL). After centrifugation for 15 minutes at 625g, the broad interphase between the Ficoll layer and the medium was collected and washed twice with washing buffer and finally washed once with Ham's F-10 medium supplemented with 5% FCS. Mononuclear cells were usually cultured in the latter medium. Flow cytometric analysis using FITC-labeled CD45/CD14 antibodies (Dianova-Immunotech) indicated that 54±17% of mononuclear cells were monocytes. In some experiments, monocytes were further purified by incubating the PBMC suspension for 15 minutes at 4°C with microbeads conjugated to anti-human CD14 antibodies and thereafter passaged over a positive selection column (Miltenyi Biotech). Thereby a suspension consisting of 98% monocytes was obtained. The monocytic cell line Mono Mac-618 (kindly provided by Dr Löms Ziegler-Heitbrock, University of Munich) was cultured in Ham's F-10 medium supplemented with 5% FCS.

Endotoxin Contamination
Ham's F-10, FCS, and NaCl-Tris buffer were routinely tested for endotoxin contamination with the Coatest kit (Chromogenix). The endotoxin concentration of the media and buffer was always <0.1 ng/mL.

Pretreatment of Cells With Lysophospholipids
Usually, 106 PBMCs in 1 mL of Ham's F-10 medium supplemented with 5% FCS were preincubated at 37°C with different concentrations of 1-palmitoyl-2-lysoPC or either lysophosphatidylethanolamine (lysoPE), lysophosphatidylserine (lysoPS), or lysophosphatidylinositol (lysoPI; all obtained from Sigma) dissolved in 0.5% to 1% ethanol. Trypan blue exclusion indicated that >95% of the cells were viable after incubation with 50 µmol/L of lysoPC, lysoPE, lysoPS, or lysoPI. Subsequent to the preincubation, the suspension was centrifuged for 10 minutes at 175g and cells resuspended for incubation with LPS.

Phospholipid Analysis of Monocytes
The PBMC suspension was incubated with 10 µmol/L lysoPC at 37°C or with 1% ethanol. After centrifugation and removal of the supernatant, the cell pellet was resuspended in Ham's F-10 medium (with 5% FCS), and monocytes were isolated by using microbeads conjugated with CD14 antibodies as detailed above. Lipids from 1 mL of monocyte suspension containing 4x106 cells were extracted according to Reference 1919 . The lipid extract was applied to silica G60 plates (Merck), previously sprayed with potassium oxalate (1% in methanol and water, 2:3 vol/vol), and activated for 30 minutes at 80°C. The plates were developed in the first dimension with the solvent system chloromethane/methanol/ammonia/water (45:37:6:4, vol/vol/vol/vol); dried; and in the second dimension, treated with chloromethane/methanol/acetone/acetic acid/water (40:15:15:12:8, vol/vol/vol/vol/vol). For quantification of phospholipids, the phosphate contents of the spots were determined as previously detailed.20

Assessment of TF Activity
After preincubation with lysophospholipids (or vehicle), PBMCs or Mono Mac-6 cells (105/well) were incubated for 6 hours at 37°C in the presence of LPS (from Escherichia coli or Salmonella minnesota) in 96-well plates with 200 µL of Ham's F-10 medium (without phenol red) containing 5% FCS. Subsequently, the medium was removed, deoxycholate (0.15%, vol/vol) was added, and the cells were subjected to 2 cycles of freezing and thawing. TF activity was assayed essentially as described previously21 by a 1-stage amidolytic assay using the chromogenic substrate S2222. The cell lysate was incubated for 25 minutes at 30°C in the above-specified Ham's F-10 medium containing 0.88 U/mL factor VII (final concentration) of a coagulation factor concentrate consisting of factors II, VII, IX, and X (Beriplex) and 125 µg/mL (final concentration) of the chromogenic substrate S2222 (Chromogenix). The increase in optical density at 405 nm was monitored in an ELISA reader (Dynatech MR 7000, Dynatech Laboratories). A standard curve was prepared by using dilutions of TF concentrates (Thromborel S from human placenta; Behring).

Determination of TF Antigen
After preincubation of the PBMCs with lysophospholipids and subsequent incubation in the presence of LPS, the mononuclear cells were separated from the incubation buffer by centrifugation, and the supernatant was stored for determination of TF antigen released into the extracellular medium. The cells were disrupted by a 3-second sonication on ice, and cell fragments were extracted for 12 hours at 4°C with a buffer composed of 100 mmol/L NaCl, 50 mmol/L Tris (pH 7.49), and 0.1% Triton X-100. After a 10-minute centrifugation, TF antigen in the cell extracts was measured by using a commercially available kit according to the instructions of the manufacturer (Imubind TF ELISA Kit, Loxo GmbH). The method uses a monoclonal antibody against the TF antigen coupled to a biotinylated rabbit polyclonal antibody for detection.

Assessment of LPS Binding and Internalization
Subsequent to preincubation with lysoPC or vehicle, the mononuclear cell suspension was washed once with Ham's F-10 containing 5% FCS and resuspended in the same medium. R7-LPS (from S minnesota) was added, and the suspension was incubated further for 60 minutes at 4°C. Binding of R7-LPS was detected by using a primary anti–R7-LPS monoclonal antibody (clone S3232) coupled to a secondary FITC-labeled goat anti-mouse antibody. For estimation of LPS internalization, after a 60-minute incubation of mononuclear cells at 4°C in the presence of R7-LPS, the suspension was further incubated for 360 minutes without LPS at either 4°C or 37°C. From the differences in fluorescence parameters at the 2 temperatures, the amount of LPS internalized can be estimated, because LPS is incorporated at 37°C but not at 4°C and the LPS taken up by the cells becomes inaccessible to the anti-LPS antibody. Selective LPS binding and internalization into monocytes were determined in a Cytofluorograf (system 50H, Ortho Diagnostic Systems Inc) through gating in the forward/sideward scatter.

Determination of cAMP Content
Monocytes were isolated from the mononuclear cell suspension by using CD14 antibodies (see above) and thereafter preincubated for 60 minutes at 37°C with lysoPC. Subsequently, the cells were resuspended in a medium containing 50 mmol/L Trizma, 20 mmol/L NaCl, 5 mmol/L KCl, 9.8 mmol/L MgCl2, 2.7 mmol/L Na2EDTA, and 0.1 mmol/L 3-isobutyl-1-methylxanthine (pH 7.4) and incubated for 15 minutes at 37°C. After centrifugation for 10 minutes at 6000g and 4°C, the supernatant was analyzed for cAMP content by using a kit according to the instructions of the manufacturer (Amersham-Buchler).

Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts from PBMCs were isolated and analyzed as previously described.22 Protein concentrations were determined by the Bradford method (Bio-Rad). Oligonucleotides with a {kappa}B consensus motif or with the {kappa}B-like site of the TF promoter were used as probes and labeled by annealing of complementary primers. This process was followed by primer extension with the Klenow fragment of DNA polymerase I (Boehringer Mannheim) in the presence of [{gamma}-32P]dCTP (>3000 Ci/mmol; DuPont) and deoxynucleoside triphosphates (Boehringer Mannheim). Nuclear extracts (5 µg protein) were incubated with radiolabeled DNA probes ({approx}10 ng; 105 cpm) for 30 minutes at room temperature in 20 µL of binding buffer (20 mmol/L Tris-HCl, 50 mmol/L KCl, 1 mmol/L DTT, 0.5 mmol/L EDTA, 1 mg/mL albumin, 5% glycerol, 0.2% NP-40, and 50 ng/µL of poly[dI-dC], pH 7.9). Samples were run in 0.25x TBE buffer (10x TBE is 890 mmol/L Tris, 890 mmol/L boric acid, and 20 mmol/L EDTA, pH 8.0) on nondenaturing 4% polyacrylamide gels at 125 V. As a control, samples were treated with a 100-fold excess of nonlabeled {kappa}B oligonucleotide, which completely abolished binding of the radiolabeled oligonucleotide to the nuclear proteins. The binding of transcription factor Sp1 was analyzed by EMSA by using a specific consensus oligonucleotide labeled with [{gamma}-32P]ATP (>5000 Ci/mmol; DuPont) and T4 polynucleotide kinase (Boehringer Mannheim). After the gels were dried, they were analyzed by autoradiography.

Northern Blot Analysis
Total RNA was extracted from PBMCs by using either the microRNA isolation kit (Stratagene) or RNA Instapure (Eurogentec). Total RNA (5 µg) was electrophoresed through a denaturing 1.2% formaldehyde gel and capillary-blotted overnight onto a nylon membrane (Hybond-N, Amersham). Hybridization was carried out overnight at 42°C by using randomly primed cDNA probes (Multiprime, Amersham). The blots were washed with increasingly stringent concentrations of SSC at 52°C and exposed to autoradiography films. The cDNA probe utilized has been described previously.23 To control for variability in sample loading, the blots were rehybridized with a 500-bp EcoRI fragment of GAPDH cDNA.

Statistical Analysis
Statistical analysis was performed by 1-way ANOVA or by Student's paired t test where appropriate. Values of P<0.05 were considered significant.


*    Results
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 up arrowMethods
 *Results
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Mononuclear cells suspended in Ham's F-10 medium supplemented with 5% FCS were incubated for different time intervals at 37°C with 0.5 to 1000 ng/mL LPS. Control experiments indicated optimal stimulation of TF activity in the presence of 10 ng/mL LPS (from either E coli or S minnesota) after 6 hours of incubation, no further increase in activity being observed at longer incubation intervals or with higher concentrations of the LPS (data not shown). In mononuclear cells treated for 6 hours with 10 ng/mL LPS, TF activity was elevated by 8.3-fold compared with monocytes incubated without LPS (Figure 1Down, upper panel).



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  		Figure 1. TF activity and TF antigen expression of mononuclear cells is specifically inhibited by lysoPC. Mononuclear cells suspended in Ham's F-10 medium supplemented with 5% FCS were preincubated for 60 minutes at 37°C with the indicated concentrations (in µmol/L) of lysoPC, lysoPE, or vehicle. Thereafter, the cells were washed once and incubated for another 6 hours with LPS (10 ng/mL) at 37°C in Ham's F-10 medium containing 5% FCS. Subsequently, monocytic TF activity (upper panel) was assessed by a 1-stage amidolytic assay as described in Methods. TF antigen expression (lower panel) was determined by using a monoclonal antibody against TF (see Methods). Column (col) 1, control (-LPS); col 2, +LPS; col 3, +1 µmol/L lysoPC+LPS; col 4, +2.5 µmol/L lysoPC+LPS; col 5, +5 µmol/L lysoPC+LPS; col 6, +10 µmol/L lysoPC+LPS; col 7, +20 µmol/L lysoPC+LPS; col 8, +50 µmol/L lysoPC+LPS; col 9, +1 µmol/L lysoPE+LPS; col 10, +2.5 µmol/L lysoPE+LPS; col 11, +10 µmol/L lysoPE+LPS; and col 12, +50 µmol/L lysoPE+LPS. Values are mean±SD of experiments on 6 through 10 different mononuclear cell preparations. *P<0.05 versus col 2 by 1-way ANOVA.

Preincubation of PBMCs with 1 to 50 µmol/L 1-palmitoyl-2-lysoPC for 60 minutes dose-dependently lowered the stimulatory effect of LPS on TF activity. With 10 and 20 µmol/L lysoPC, TF activity induced by LPS was decreased by 73% and 71%, respectively, no further reduction being evident at 50 µmol/L (Figure 1Up, upper panel). Basal TF activity was not influenced by preincubation with 10 µmol/L lysoPC (not shown). LPS-induced TF activity was inhibited to a similar degree in monocytes preincubated for 30 minutes with 10 µmol/L lysoPC after a 6-hour stimulation with 10 ng/mL LPS from 3.1±1.2 mU/106 cells (without lysoPC) to 1.3±0.4 mU/106 cells (with lysoPC). 1-Palmitoyl-2-lysoPE, a lysophospholipid species differing from lyso PC by the lack of 3 methyl groups, was added to the preincubation medium in the concentration range 1 to 50 µmol/L. LPS-induced TF activity was unaffected by all concentrations of lysoPE investigated (Figure 1Up, upper panel). Similarly, a 60-minute preincubation with 50 µmol/L of either lysoPS or lysoPI barely altered TF activity as elicited by LPS (data not shown).

To analyze the effect of lysoPC on TF expression at the protein level, TF antigen was determined in the mononuclear cell suspension by use of a monoclonal anti-TF antibody (see Methods). LPS (10 ng/mL) increased TF antigen expression by 5.3-fold in the absence of added lysoPC (Figure 1Up, lower panel). After preincubation with 10 or 50 µmol/L lysoPC, the antigen expression elicited by LPS was inhibited by 76% or 80%, respectively. After preincubation with 50 µmol/L lysoPE, the stimulated TF antigen expression was barely changed (Figure 1Up, lower panel). Together, the data of Figure 1Up indicate that the inhibition of TF activity by lysoPC is most probably due to a reduction in the amount of TF protein.

In the experiments shown in Table 1Down, phospholipids of monocytes were quantified before and after a 60-minute incubation with 10 µmol/L lysoPC. PC, PE, sphingomyelin, PS, lysoPC, and PI were the major phospholipids of monocytes, in accordance with earlier work.24 The content of monocytic lysoPC was increased by 158% after preincubation with lysoPC, whereas the contents of the other phospholipids analyzed were unchanged. Thus, preincubation with lysoPC results in a selective increase of the lysoPC content of monocytes.


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  		Table 1. Selective Increase in LysoPC Contents of Human Monocytes After Preincubation With LysoPC

It can be safely assumed that the determinations of TF activity and expression shown in the experiments of Figure 1Up are exclusively related to the action of monocytic TF because other blood cells do not express the protein.25 Conceivably, however, the effect of lysoPC on monocytic TF activity could be mediated by other blood cells (lymphocytes, platelets) present in the cell preparation. Therefore, experiments were conducted with the monocytic cell line Mono Mac-6. Mono Mac-6 cells cultivated in Ham's F-10 containing 5% FCS were preincubated with different concentrations of lysoPC and thereafter challenged with 100 ng/mL LPS for 6 hours. As can be deduced from Figure 2Down, increasing the lysoPC concentration from 2.5 to 20 µmol/L dose-dependently lowered the LPS-induced TF activity of the cells. A 72% and 71% inhibition was noticed at 20 and 50 µmol/L of the lysophospholipid, respectively. Preincubation with 50 µmol/L lysoPE did not alter TF activity as induced by LPS (not shown). Thus, the effect of lysoPC is most probably independent of the presence of nonmonocytic cells.



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  		Figure 2. TF activity of Mono Mac-6 cells is suppressed by lysoPC. Mono Mac-6 cells cultivated in Ham's F-10 medium containing 5% FCS were incubated for 60 minutes at 37°C with the indicated concentrations of lysoPC (in µmol/L). Subsequently, the cells were washed once and incubated for another 6 hours at 37°C with LPS (100 ng/mL). Column (col) 1, control (-LPS); col 2, +LPS; col 3, +2.5 µmol/L lysoPC+LPS; col 4, +5 µmol/L lysoPC+LPS; col 5, +10 µmol/L lysoPC+LPS; col 6, +20 µmol/L lysoPC+LPS; and col 7, +50 µmol/L lysoPC+LPS. Values are mean±SD of experiments on 5 different cell preparations. *P<0.05 vs col 2 by 1-way ANOVA.

A substantial portion of the lysoPC incorporated into the monocytes after preincubation with the lysophospholipid is expected to be localized in the plasma membrane. Therefore, lysoPC could inhibit LPS-induced TF expression by altering the interaction of LPS with LPS-binding sites on the plasma membrane of monocytes. Control experiments indicated that binding of LPS to the monocytes was saturated after 1 hour of incubation at 4°C. The amount of LPS bound to monocytes at this time point was completely unaffected by preincubation with 5, 10, or 50 µmol/L lysoPC (Table 2Down). This excludes the possibility that the inhibition of TF expression by lysophospholipid was related to an effect on LPS binding. In further experiments, monocytes (pretreated for 60 minutes with either 10 µmol/L lysoPC or ethanol) were first incubated for 60 minutes at 4°C with LPS and thereafter for an additional 6 hours at either 4°C or 37°C (in the absence of LPS). The differences between the fluorescence intensities obtained at the 2 temperatures after the final 6-hour incubation period were used to estimate LPS internalization (see Methods). These differences were not affected by pretreatment with lysoPC (Table 2Down).


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  		Table 2. LPS Binding and Internalization Into Human Monocytes Is Not Affected by LysoPC

Induction of TF expression of monocytes is regulated at both the transcriptional and posttranscriptional level.22 23 Activation of p65/c-Rel complexes has been implicated in the LPS-induced expression of TF.26 Therefore, EMSAs were performed with a labeled {kappa}B consensus oligonucleotide and an oligonucleotide containing the specific {kappa}B-like site identified in the TF promoter.22 26 Treatment of mononuclear cells with LPS for 60 minutes strongly increased binding activity to both sites (Figure 3Down, upper and lower panels). This effect was markedly inhibited when cells had been pretreated for 60 minutes with lysoPC (10 µmol/L). No effect was seen after pretreatment with lysoPE. Binding to an Sp-1 oligonucleotide was not affected by lysoPC. Northern blot analyses of steady-state TF mRNA levels were performed after a 2-hour incubation of mononuclear cells in the presence or absence of LPS. The TF mRNA expression elicited by LPS was partially reduced after pretreatment of the cells with lysoPC (Figure 4Down). In a total of 3 experiments (including the 1 shown in Figure 4Down), LPS-induced TF mRNA expression was lowered by 38±10% by lysoPC pretreatment, the effect not reaching statistical significance (versus LPS-mediated TF mRNA expression without lysoPC; paired t test).



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  		Figure 3. LysoPC inhibits NF-{kappa}B activation. Mononuclear cells suspended in Ham's F-10 medium supplemented with 5% FCS were preincubated for 60 minutes at 37°C with lysoPC, lysoPE (10 µmol/L each), or vehicle. Thereafter, the cells were washed once and incubated for a further 1 hour (NF-{kappa}B) with LPS (10 ng/mL) at 37°C in Ham's F-10 medium containing 5% FCS. Nuclear extracts were analyzed by EMSA using oligonucleotides with the {kappa}B-like site specific for the TF promoter (upper panel) or a {kappa}B consensus sequence (lower panel). Binding of nuclear proteins to an oligonucleotide containing the Sp-1 consensus sequence was examined in the same extracts. Representative results of experiments on 3 through 5 different cell preparations are shown.



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  		Figure 4. TF mRNA expression is reduced after pretreatment of mononuclear cells with lysoPC. Mononuclear cells suspended in Ham's F-10 medium supplemented with 5% FCS were preincubated for 60 minutes at 37°C with lysoPC or vehicle. Thereafter, the cells were washed once and incubated for an additional 2 hours with LPS (10 ng/mL) at 37°C in Ham's F-10 medium containing 5% FCS. Nuclear extracts were analyzed for TF and GAPDH mRNA expression as described in Methods. The densitometric analysis of TF mRNA as normalized to GAPDH mRNA is shown in the lower part of the figure.

Previous data indicate that induction of TF expression in different cell types is inhibited by increases in intracellular cAMP.27 28 The potential role of intracellular cAMP in the inhibition of TF activity elicited by lysoPC was therefore evaluated. In a first set of experiments, the effect of the lysophospholipid on monocytic cAMP levels was analyzed. Monocytes were isolated from mononuclear cell suspensions by using anti-CD14 antibodies (see Methods). The cells (106) were incubated for 60 minutes in Ham's F-10 medium (supplemented with 5% FCS) in the presence of either lysoPC (20 µmol/L) or ethanol (1%). Thereafter, intracellular cAMP levels were estimated by using a kit as detailed in Methods. In monocytes pretreated with lysoPC, cAMP levels were raised by 64% (10 µmol/L lysoPC) and 72% (20 µmol/L lysoPC) compared with cells pretreated with vehicle (Table 3Down).


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  		Table 3. LysoPC Pretreatment Increases the cAMP Contents of Human Monocytes

In further experiments, 2'-deoxy-3'-adenosine monophosphate (dAMP, 0.5 mmol/L), an inhibitor of adenylyl cyclase, was added to the mononuclear cell suspension 90 minutes before the start of incubation with LPS. The stimulation of TF activity elicited by LPS was not affected by dAMP (Figure 5Down, columns 1 through 3). In monocytes pretreated with 10 µmol/L lysoPC, LPS-induced TF activity was inhibited by 72% (column 4). In the presence of dAMP, this inhibition was partially reversed (column 5). Furthermore, the cell suspension was incubated with dibutyryl cAMP (1 mmol/L), a membrane-permeable analogue of cAMP. Therefore, the LPS-induced TF activity was abolished both in untreated cells as well as in monocytes pretreated with lysoPC (columns 6 and 7).



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  		Figure 5. Agents modifying intracellular cAMP levels differentially affect the TF activity of lysoPC-pretreated mononuclear cells. Mononuclear cells suspended in Ham's F-10 medium supplemented with 5% FCS were preincubated for 60 minutes at 37°C with lysoPC (10 µmol/L) or vehicle. Thereafter, the cells were washed once and incubated for another 6 hours with LPS (10 ng/mL) at 37°C in Ham's F-10 medium containing 5% FCS. To decrease intracellular cAMP levels, dAMP (0.5 mmol/L) was added 90 minutes before the start of the incubation with LPS. To elevate intracellular cAMP levels, dibutyryl cAMP (1 mmol/L) was added to the cell suspension together with LPS. Monocytic TF activity was assessed by a 1-stage amidolytic assay as described in Methods. Column (col) 1, control (-LPS); col 2, +LPS; col 3, +dAMP+LPS; col 4, +lysoPC+LPS; col 5, +dAMP+lysoPC+LPS; col 6, +dibutyryl cAMP+LPS; col 7, +lysoPC+dibutyryl cAMP+LPS. Values are mean±SD of experiments on 4 through 6 different mononuclear cell preparations. *P<0.05 col 4 through 7 versus col 2; col 4, 6, and 7 versus col 5.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
We observed in the present study that preincubation with lysoPC suppressed LPS-induced TF expression and activity of monocytic cells. The inhibition exerted by lysoPC was selective, because other lysophospholipids were ineffective (Figures 1Up and 3Up and Results). The effect of lysoPC on LPS-induced TF expression was apparently related to the presence of lysoPC within the monocytes. Indeed, preincubation with the lysophospholipid induced an increase in lysoPC contents of monocytes without inducing major changes in the levels of other phospholipids (Table 1Up).

To clarify the mechanisms mediating inhibition of TF activity by lysoPC, we first evaluated whether the lysophospholipid affected the binding of LPS to the extracellular surface of the monocytes. These cells are known to possess plasma membrane–binding sites for LPS, such as CD14 receptors, for example.29 Preincubation with lysoPC neither influenced the interaction of LPS with its binding sites on the cell membrane of monocytes nor affected LPS internalization (Table 2Up). This result indicated that lysoPC interfered further downstream in the LPS-elicited signaling cascade.

According to present knowledge, LPS-mediated TF gene expression is transcriptionally regulated by members of the nuclear factor (NF)-{kappa}B/Rel family, AP-1 and Sp1.22 23 30 Activation of NF-{kappa}B/Rel was potently inhibited by the lysophospholipid (Figure 3Up). A recent report indicates that lysoPC at concentrations similar to those exerting a maximal response in the present study stimulates nuclear binding and transcriptional activity of AP-1.31 The stimulation of AP-1 activity by lysoPC may counteract the inhibitory effect of lysoPC on NF-{kappa}B/Rel activity. This process might explain why LPS-induced TF mRNA levels tended toward only partial reduction in the presence of lysoPC (Figure 4Up).

Several previous investigations reported inhibition of TF expression by agents known to increase intracellular cAMP levels.27 28 Furthermore, intracellular cAMP contents were shown to be increased by lysoPC in platelets and THP-1 cells.32 We therefore investigated whether intracellular cAMP was involved in the effect of lysoPC on the TF activity of monocytes. Preincubation of the mononuclear cell suspension with dAMP, an inhibitor of adenylyl cyclase, partially reversed the inhibition of LPS-induced TF activity elicited by lysoPC (Figure 5Up). LysoPC pretreatment led to an increase in monocytic cAMP levels (Table 3Up). These results argue in favor of the hypothesis that inhibition of LPS-induced TF activity induced by lysoPC is in part mediated by an increase in intracellular cAMP. Inhibition of NF-{kappa}B/Rel activity in lysoPC-pretreated monocytes (Figure 3Up) is in agreement with this hypothesis, as increases in intracellular cAMP were shown to block the activities of these transcription factors in several investigations.33 34 35 A recent study indicates that although elevations in intracellular cAMP levels resulted in inhibition of NF-{kappa}B/Rel–mediated transcription of several genes (including TF), nuclear binding activities of these factors were unchanged.36 The basis for these differences is unknown at present.

LPS-promoted monocytic TF expression was clearly diminished by lysoPC (Figures 1Up and 2Up). TF mRNA expression tended to be partially lowered (Figure 4Up). Together, these results could indicate that posttranscriptional mechanisms are also involved in the inhibitory effect of lysoPC on TF expression. Two recent reports underline the importance of posttranscriptional processes for the inhibition of TF expression.37 38 In 1 of those studies,37 pyrrolidine dithiocarbamate, a strong inhibitor of NF-{kappa}B activation, was found to inhibit TF expression without inducing considerable alterations in TF mRNA levels. A somewhat similar situation was encountered in the present study in human monocytes preincubated with lysoPC.

In vitro oxidized LDL has been shown to alter activation of NF-{kappa}B/Rel complexes, the effect depending on the degree of oxidation.39 40 Whereas mildly oxidized LDL particles stimulated the activities of NF-{kappa}B/Rel, strongly oxidized LDL particles were shown to inhibit activation of the transcription factors. The lysoPC contents of LDL increase with increasing strength of oxidation.16 Accordingly, the inhibitory action of heavily oxidized LDL on activation of NF-{kappa}B/Rel could probably be caused by elevations in LDL-associated lysoPC.

Stimulation of TF synthesis has been proposed to be responsible for the thromboembolic complications associated with inflammation and atherosclerosis.1 As outlined in the introduction, under these conditions increased levels of lysoPC are frequently observed as a result of activation of PLA2. LysoPC appears to promote a variety of inflammatory and atherogenic responses in cellular components of the vascular bed, such as increased adhesion of monocytes to the endothelium13 or activation of distinct growth factors contributing to proliferation of endothelial cells, for example.14 However, the influence of lysoPC on the expression of several endothelial proteins involved in inflammation and atherogenesis clearly differed from the effects exerted by inflammatory cytokines such as tumor necrosis factor-{alpha}.

Furthermore, lysoPC has recently been shown to increase the synthesis of endothelial prostacyclin, a potent inhibitor of platelet aggregation.41 This lysophospholipid was also shown to directly block platelet aggregation and the synthesis of platelet thromboxane A2 as elicited by platelet agonists.32 According to the results of the present study, LPS-induced expression of TF is markedly diminished in the presence of lysoPC. When monocytes are activated during inflammation, TF expression is increased. Under the same conditions, lysoPC is generated by several cells present in the blood and the vascular wall. This event, in turn, might contribute to mitigate initiation of coagulation of the inflammatory process. Some recent data point to a decisive role for macrophage TF expression in the thrombogenicity of the atherosclerotic plaque.7 8 9 The high levels of lysoPC in the plaques42 are expected to reduce TF expression of macrophages. Taken together, the results of the present and the above-mentioned investigations indicate that lysoPC, while promoting several inflammatory and atherogenic cellular responses, may concomitantly attenuate activation of coagulation.


*    Acknowledgments
 
This study was supported by grants from the Wilhelm Sander-Stiftung and the Friedrich-Baur-Stiftung to B.E. and from the Deutsche Forschungsgemeinschaft to K.B. (Br 1026/3-1 and SFB 469). The excellent technical assistance given by Tamara Eisele and Sylvia de Jonge is gratefully acknowledged. Thanks are also due to Andreas Brosig who participated in the initial phase of the experiments. We are grateful to Dr Nigel Mackman for helpful suggestions.

Received February 25, 1998; accepted May 7, 1998.


*    References
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