© 1999 American Heart Association, Inc.
Original Contributions |
Tissue Factor Expression of Human Monocytes Is Suppressed by Lysophosphatidylcholine
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
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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-
B/Rel to a TF-specific 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-B
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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-
(TNF-), 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.
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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 B consensus motif or
with the 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
[
-32P]dCTP (>3000 Ci/mmol; DuPont) and
deoxynucleoside triphosphates (Boehringer Mannheim). Nuclear
extracts (5 µg protein) were incubated with radiolabeled DNA probes
(
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 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
[-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.
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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 1
, upper panel).
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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 1
, 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 1, 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 1, 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 1, lower panel). Together, the data of Figure 1
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 1,
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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It can be safely assumed that the determinations of TF activity and
expression shown in the experiments of Figure 1 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 2, 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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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 2). 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 2).
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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 B consensus oligonucleotide and an
oligonucleotide containing the specific 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 3, 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 4). In a total of 3 experiments
(including the 1 shown in Figure 4), 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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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 3).
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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 5, 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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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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 1
and 3 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 1).
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 2). 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)-B/Rel family, AP-1 and Sp1.22 23 30
Activation of NF-B/Rel was potently inhibited by the
lysophospholipid (Figure 3). 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-B/Rel activity. This process might explain why
LPS-induced TF mRNA levels tended toward only partial reduction in the
presence of lysoPC (Figure 4).
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 5). LysoPC pretreatment led to an increase in monocytic cAMP
levels (Table 3). 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-B/Rel activity in lysoPC-pretreated monocytes (Figure 3) 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-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 1 and 2). TF mRNA expression tended to be partially lowered
(Figure 4). 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-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-B/Rel
complexes, the effect depending on the degree of
oxidation.39 40 Whereas mildly oxidized LDL
particles stimulated the activities of NF-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-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-.
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.
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Acknowledgments |
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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.
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