© 1997 American Heart Association, Inc.
Articles |
Modified LDL Decreases the Binding of Prostaglandin E2, I2, and E1 Onto Monocytes in Patients With Peripheral Vascular Disease
From the Departments of Nuclear Medicine (S.R.L., A.K., C.B., M.L., I.V.), Physiology (E.K.), Cardiology (F.R.), and Gastroenterology (J.P.), University of Vienna, A-1090 Vienna, Austria.
Correspondence to Irene Virgolini, MD, Department of Nuclear Medicine, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
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Abstract |
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Abstract Recent data suggest that various eicosanoids including prostaglandins play an important regulatory role in the development of atherosclerotic lesions. Peripheral blood monocytes have been implemented in early atherogenesis because they express receptors specific for modified LDL. In this study we investigated the binding of tritium prostaglandins E2 (3H-PGE2), E1 (3H-PGE1) and I2 (3H-PGI2) onto intact peripheral monocytes isolated from 20 patients (32-71 years) with manifested ischemic peripheral vascular disease stage II according to Fontaine and compared the results with those obtained in 16 healthy volunteers (21-68 years). In control subjects, Scatchard analyses of the binding data indicated a single class of high-affinity binding sites for 3H-PGE2 (maximal binding capacity [Bmax]=11400±3200 sites/cell; dissociation constant [Kd]=1.3±0.5 nmol/L) and two classes of binding sites for 3H-PGE1 (Bmax1=11200±4900 sites/cell, Kd1=1.5±0.5 nmol/L; Bmax2=47800±6100 sites/cell, Kd2=12.8±5.9 nmol/L) as well as for 3H-PGI2 (Bmax1=10100±3700 sites/cell, Kd1=1.7 ±0.7 nmol/L; Bmax2=81200±5200 sites/cell, Kd2=14.2±6.5 nmol/L). In the patients, an absence of the higher-affinity binding class and significantly (P<.01) fewer lower-affinity binding sites were found for each ligand (PGE2: Bmax= 6600±3600 sites/cell, Kd=12.1±3.2 nmol/L; PGI2: Bmax=6400 ±3100 sites/cell, Kd=22.1±8.3; PGE1: Bmax=5300±1700 sites/cell, Kd=20.5±7.0 nmol/L). After incubation of monocytes with modified LDL (oxidized LDL or acetylated LDL), the binding of prostaglandins was significantly (P<.01 to P<.001) decreased, whereas native VLDL, LDL, and HDL did not interfere with prostaglandin binding. Prostaglandin-induced adenosine 3'-5' cyclic monophosphate (cAMP) formation by monocytes was significantly (P<.01) lower in patients (the concentrations causing 50% elevation of basal cAMP formation [ED50] were 3.8±2.4 nmol/L for PGE2, 6.3±3.5 nmol/L for PGE1, and 5.6±4.1 nmol/L for PGI2) than in the control subjects (ED50 was 1.6±1.2 nmol/L for PGE2, 4.8±2.5 nmol/L for PGE1, and 3.1±1.4 nmol/L for PGI2). After preincubation with modified LDL, the PG-induced cAMP production by monocytes was remarkably decreased in both patients and control subjects (P<.05). Our results suggest a direct effect of modified LDL on PGE2, PGE1, and PGI2 binding onto monocytes by reducing the number of cell surface–expressed receptors available. Modified LDL also reduces the sensitivity of monocytes to prostaglandins, which results in decreased cAMP production. The complex interactions between prostaglandins and lipoproteins may play an important role during atherogenesis.
Key Words: LDL • HDL • monocytes • modified LDL • arteriosclerosis
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Introduction |
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Blood monocytes and macrophages play an important role in the pathogenesis of atherosclerosis. Atherosclerotic plaques are rich in foam cells which are believed to stem either from resident macrophages of the arterial wall, or from circulating blood monocytes by penetrating through the endothelial lining of the artery and migrating into the subendothelial space.1 2 Recently, uptake of modified LDL by the scavenger receptor expressed on monocytes has been shown to be critical for foam cell formation.3 4
Monocytes and macrophages have also been shown to produce various AA metabolites including PGE2, PGI2, and thromboxane A2.5 6 The eicosanoids may, in turn, modulate monocyte and macrophage cell functions.7 To exert their regulatory functions, prostaglandins bind to specific cell surface receptors, which have been described for several tissues and various cell types8 9 10 11 including monocytes and macrophages.12 13 14 15 Recent evidence suggests that prostaglandins may interfere with the binding of lipoproteins to lipoprotein receptors in vitro16 as well as in vivo.17 However, until now, these findings have not been related to human atherosclerotic disease. Furthermore, to our knowledge, the interaction of plasma lipoproteins with the binding of prostaglandins to monocytes and macrophages has so far not been investigated. We studied the binding of PGE2, PGE1, and PGI2 onto monocytes obtained from patients with manifested peripheral vascular disease and healthy volunteers. In a further step, the influence of various lipoproteins on the binding of prostaglandins to monocytes as well as on cAMP formation was examined.
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Methods |
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Subjects
Twenty patients (12 men and eight women; age range, 32-71 years) with peripheral vascular disease in Fontaine stage II and 16 healthy volunteers (eight men and eight women; age range, 21-68 years) were investigated. Neither the patients nor the healthy control subjects had taken any medication known to interfere with the prostaglandin system or lipid metabolism 3 weeks before and during the study. None of the healthy volunteers was a smoker or had any other known risk factor for the development of atherosclerosis. The study was performed according to the Declaration of Helsinki II. All patients were nonsmokers, but were affected by hyperlipoproteinemia type IIa.
Isolation of Monocytes
Mononuclear cells were isolated and purified from
peripheral blood using the method described by
Boyum.18 Anticoagulated blood was diluted with an equal
volume of balanced salt solution and carefully layered over Ficoll's
medium (density, 1.077 g/mL). Following
centrifugation for 25 minutes at 500g and
22°C, the mononuclear cell fraction was gathered from the interphase
and diluted 1:2 with divalent calcium–and divalent magnesium–free PBS
containing 2 mmol/L of EGTA, pH 7.4. The cells were washed
twice in elutriation medium (divalent calcium–and divalent
magnesium–free PBS, 0.5 mmol/L EGTA, pH 7.4) and
thereafter resuspended in 3 mL RPMI 1640 medium. Monocytes were then
isolated by centrifugal elutriation using a JE6 elutriator rotor in a
J6-M refrigerated centrifuge (Beckman Instruments, Inc, Palo
Alto, Calif). About 1.5x108 mononuclear cells were used
for elutriation centrifugation. The flow rate was kept
constant (15 mL/minute) and the rotor speed was reduced stepwise from
3000 to 2000 rpm. Monocytes were collected into 50-mL fractions at a
speed of approximately 2500 to 2100 rpm (variable from one patient
to the other), and kept on ice. The purity of the monocyte preparations
used was greater than 90% as judged morphologically on Giemsa staining
and with peroxidase and esterase histochemistry, as well as with
fluorescence-activated cell sorter analysis
using the anti-Leu-M3 antibody (Becton Dickinson & Co, Mountain View,
Calif). Cellular viability was higher than 96% as judged with
Trypan-blue and microscopy.
Isolation and Preparation of Lipoproteins
LDL (density, 1.019-1.063 g/mL) and HDL (density,
1.063-1.210 g/mL) were isolated from 16 normolipemic blood
donors and were prepared from the fresh plasma by sequential
ultracentrifugation using potassium bromide for density
adjustment as described previously in detail.19 The
lipoproteins were subjected to dialysis against PBS, pH 7.4, containing
0.1 mg/mL EDTA, stored at 4°C and used within 1 week.
Lipoproteins were concentrated by ultrafiltration using Centrisart
membranes (Sartorius, Göttingen, Germany). Acetylated LDL
was prepared by adding saturated sodium acetate solution to 4 mg of
protein per milliliter of LDL (vol:vol=1:1) under continuous stirring
at 4°C.20 Acetic anhydride was added over a period of 1
hour with regard to total lysines in LDL. Extensive dialysis was
performed on the modified LDL against saline EDTA. The modification was
determined by titration of free amino group with TNBS21
and electrophoresis.22 Oxidized LDL was obtained by
dialysis of LDL against PBS overnight, followed by incubation of LDL
(0.4 mg/mL LDL protein) with copper chloride (10
µmol/L) at 37°C for 20 hours.23 The oxidation
was stopped by adding EDTA (1 mmol/L), which was removed by
chromatography on Sephadex G25 (Pharmacia, Sweden)
before application in the binding assays. The degree of LDL oxidation
was monitored by determining malondialdehyde equivalents with
TBARS24 and electrophoresis.22 Lipid
hydroperoxides of LDL were measured at 365 nm by their capacity to
convert iodide to iodine.25 The lipopolysaccharide
content of modified LDL was examined by using a Limulus assay kit
(Sigma Chemical Co, St Louis, Mo). Copper or Ac2O were
removed with chromatography on Sephadex G25 before cell
incubation. The total protein content of the lipoproteins was
analyzed with the method of Lowry et al.26 The
apoprotein composition of each of the lipoprotein classes was assessed
using radial immunodiffusion techniques.
The extent of acetylation of amino groups of acetylated LDL ranged from 40% to 50% as determined by TNBS reactivity. The electrophoretic mobility of acetylated LDL in agarose gels increased by 70% to 100% when compared with native LDL. Only oxidized LDL with lipid peroxide values higher than 100 nmol/mg of LDL protein and TBARS values between 15 and 20 nmol/mg of LDL protein or electrophoretic mobility increased by 80% to 110% compared with native LDL was used. The lipopolysaccharide content of modified LDL was lower than 0.1% as determined with the Limulus assay.
Prostaglandin-Binding Studies
Prostaglandin-binding experiments were carried out
with monocytes as described earlier.8 In initial studies
the binding of prostaglandin onto intact monocytes was
examined as a function of time and temperature. The specific activity
of the prostaglandin was as follows:
3H-PGE2, 142 Ci/mmol;
3H-PGE1, 45 Ci/mmol;
3H-PGI2, 14.5 Ci/mmol and the radiochemical
purity was: 3H-PGE2, 97.3%; PGE1,
98.2%; 3H-PGI2, 96.8%. (All
3H-PGs were obtained from Amersham International
[Buckinghamshire, UK]; unlabeled prostaglandins were
purchased from Upjohn [Kalamazoo, Mich]) Monocytes were suspended in
50 mmol/L Tris hydrochloric acid buffer (pH, 7.5)
containing 5 mmol/L magnesium chloride and 1
mmol/L calcium chloride and 0.1 mol/L sodium chloride as
well as 10-6 mol/L
indomethacin to prevent cells from
endogenous generation of prostaglandins.
Monocytes were then incubated with 3H-PG (10 nmol/L)
in the absence (total binding) and the presence (nonspecific binding)
of unlabeled prostaglandin (100 nmol/L),
respectively, for 2 to 120 minutes, to study the time course of
association of binding. Dissociation of binding was induced by adding
100 nmol/L unlabeled prostaglandin at different
intervals (2 to 50 minutes) at equilibrium. The dependence of
temperature on binding was studied by incubating the tubes at 4°C,
22°C, and 37°C for 45 minutes. Based on the initial results, all
further binding experiments were performed at 4°C for 45 minutes. In
saturation experiments, the intact cells were incubated with various
concentrations of 3H-PG (1 to 100 nmol/L) in the
absence (total binding) or the presence of prostaglandin
(100 nmol/L) (nonspecific binding) and incubated at 4°C for 45
minutes. Specific binding was calculated as the difference between
total and nonspecific binding. In displacement experiments the intact
cells were incubated with 5 nmol/L of 3H-PG in the
absence and the presence of various concentrations
(10-4 to 10-9
mol/L) of unlabeled prostaglandins
(PGE2, PGE1, PGI2,
PGD2, and PGF2
) as well as
17-phenyl-PGE2, butaprost, enprostil, and cicaprost
(specific EP1, EP2, EP3, and IP receptor agonists,27
respectively).
After incubation, each reaction mixture was applied to a Whatman CF/B glass filter to separate monocyte-bound from unbound 3H-PG. The filters were rinsed three times with 3 mL of 4°C Tris hydrochloric acid buffer (pH, 7.5) and the radioactivity retained by the filter was quantified in a beta scintillation counter.
cAMP Formation by Monocytes
Monocytes were washed in 50 mmol/L Tris hydrochloric
acid buffer, pH 7.5, containing 0.5 mg/mL
acetylsalicylic acid and 1 mmol/L
aminophylline, to prevent cells from endogenous
prostaglandin synthesis and degradation of cAMP,
respectively. Cells were incubated in the absence (basal cAMP
formation) or presence of various concentrations
(10-4 to 10-9
mol/L) of prostaglandins (PGE2,
PGE1, PGI2, PGD2, iloprost, and
PGF2) at 37°C for 30 minutes. The reaction
was stopped by homogenization (ultraturrax [Ika,
Germany] and ultrasound) at 4°C and centrifugation
at 5000g at 4°C for 10 minutes. The cAMP content in the
supernatant was determined with a radioimmunoassay (Amersham
International, Buckinghamshire, UK). The sensitivity of the assay
ranged from 0.25 to 16 fmol/mL.
Effect of Lipoproteins on Prostaglandin Binding to
Monocytes and cAMP-Formation
In separate experiments, the effect of native (VLDL, LDL, HDL)
and modified lipoproteins (oxidized and acetylated LDL) on
prostaglandin binding onto monocytes was investigated.
Monocytes (5x105 cells in the vial) were incubated with
the lipoproteins (100 µg/mL) for various intervals (0.15 to 3
hours) at 4°C and 37°C using various concentrations of
prostaglandins (1 to 100 nmol/L). Thereafter,
prostaglandin-binding experiments and cAMP formation were
studied as described.
Data Analyses
Binding data were calculated using the method described by
Scatchard.28 Statistical analyses were made using
standard statistical tests including Student's t test and
ANOVA at a confidence level of 95%.
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Results |
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Prostaglandin Binding onto Monocytes
In initial experiments, monocytes were incubated with 3H-PGE2, 3H-PGI2, or 3H-PGE1, respectively, to evaluate the association kinetics for receptor binding. In these studies, a rapid increase of binding within 10 minutes was observed when the monocytes were incubated with 3H-PG at 4°C. An apparent equilibrium was reached within 20 minutes. Displacement of 3H-PG could be achieved after addition of an excess of 100 nmol/L unlabeled prostaglandin (Fig 1
shows
the linear course of PGE2 binding to monocytes). The
3H-PG binding to monocytes was significantly
temperature-dependent. The binding of 3H-PGE2
at 45 minutes was higher at 4°C (96% binding) than at 22°C (92%)
and at 37°C (89%, P<.01). Therefore, all further
prostaglandin-binding experiments were carried out at 4°C
for 45 minutes. The temperature dependence of binding did not vary
considerably for the other prostaglandins.
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): 3H-PGE2 (5 nmol/L) was incubated with
monocytes in the absence (total binding) and the presence (nonspecific
binding) of unlabeled PGE2 (100 nmol/L) for the intervals
indicated. Specific binding (shown), determined as the difference of
total and nonspecific binding, reaches 95% at the ligand concentration
studied after 15 minutes. Dissociation (+): to study the time course of
displacement of specific binding (shown) an excess of unlabeled
PGE2 (100 nmol/L) was added at equilibrium (45 minutes).
3H-PGE2 is rapidly displaceable, which
indicates that only a minimal amount is incorporated under these
conditions. Each point represents the mean±SD of three
independent experiments with monocytes.
Binding of 3H-PGE2 to Monocytes
In saturation experiments, the binding of
3H-PGE2 onto monocytes from healthy control
subjects as well as patients was completely saturable (Tables 1 and 2). Scatchard analyses of the
binding equilibrium data indicated a single high-affinity binding class
that bound 11400±3200 sites/cell with a Kd of 1.3±0.5
nmol/L (Fig 2, left panel). In
patients, the 3H-PGE2–binding capacity
amounted to 6600±3600 sites/cell (Kd=12.1±3.2
nmol/L), which was significantly (P<.01) lower than
the control group (Table 1, Fig 2, right panel). The specificity of
PGE2-binding sites expressed on monocytes was studied with
competition experiments. The binding of 3H-PGE2
to monocytes was examined in the presence of unlabeled
PGE2, PGE1, PGI2,
PGF2, PGD2, butaprost,
17-phenyl-PGE2, enprostil, and cicaprost. As shown in Fig 3, unlabeled PGE2 caused
significant inhibition of 3H-PGE2 binding onto
monocytes with an IC50 of 2.0±1.1 nmol/L. Unlabeled
PGE1 (IC50=4.0±2.3 nmol/L) was
similarly effective, whereas the unlabeled selective EP3-subtype
receptor agonist enprostil (IC50=4.5±1.0 nmol/L)
and the relative selective EP1 receptor agonist
17-phenyl-PGE2 (IC50=26±13 nmol/L),
PGI2 (IC50=90±58 nmol/L), the selective
EP2 receptor agonist butaprost (IC50=430±70
nmol/L), and the selective IP receptor agonist cicaprost
(IC50=510±160 nmol/L) were less effective, and
PGF2 was ineffective
(IC50>100 µmol/L) to inhibit
3H-PGE2 binding to monocytes. The rank order of
potency was also similar in patients with peripheral
vascular disease (Table 3).
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), PGE1 (*), PGD2
(
), and PGF2a (X). The IC50 values were
2.0±1.1 nmol/L for PGE2, 4.0±2.3 nmol/L for
PGE1, 90±58 nmol/L for PGI2, 600±90 nmol/L
for PGD2 and more than 100 µmol/L for
PGF2
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Binding of 3H-PGI2 and
3H-PGE1 to Monocytes
On the surface of monocytes isolated from healthy subjects two
saturable binding classes—a low-capacity, high-affinity binding class
and a high-capacity, low-affinity binding class—were found for both
3H-PGI2 (Table 4)
and 3H-PGE1
(Table 5). Only a low-affinity binding
class for 3H-PGI2 (Table 5)
and for 3H-PGE1
(Table
6) on monocytes derived from
patients was demonstrable. As indicated in Tables 4 through 7,
the binding sites for
3H-PGI2 and 3H-PGE1 on
monocytes were significantly (P<.01) lower in patients with
peripheral vascular disease than in healthy control
subjects. 3H-PGI2 binding to monocytes was best
displaced by the selective IP receptor agonist cicaprost,
PGI2, and iloprost (IC50=11.4±5.2
nmol/L, 12.3±8.9 nmol/L, and 13.3±8.9 nmol/L,
respectively), whereas PGE1, PGE2, the
selective EP1 receptor agonist 17-phenyl-PGE2, the
selective EP3 receptor agonist enprostil, PGD2, and
PGF2 were weaker agonists
(IC50=18.5±10.6 nmol/L for PGE1,
170±100 nmol/L for PGE2, 320±70 nmol/L for
17-phenyl-PGE2, 1.5±0.3 µmol/L for
enprostil, 320±200 nmol/L for PGD2, and >100
µmol/L for PGF2). In the patients,
a similar rank order of potency was obtained (Table 3).
PGE1, 17-phenyl-PGE2, and enprostil caused
significant inhibition of 3H-PGE1 binding to
human monocytes with an IC50 value of 11.4±8.9
nmol/L, 12.7±5.6 nmol/L, 18.4±3.7 nmol/L,
respectively. Iloprost, PGI2, and PGE2 were
weaker competitors (IC50=18.5±10.7 nmol/L for
iloprost, 22±13 nmol/L for PGI2, and 19.4±10.9
nmol/L for PGE2). In the patients, the rank order of
potency was as follows: PGE1 was greater than
17-phenyl-PGE2, which was greater than PGE2,
which was greater than iloprost, which was greater than
PGI2, which was greater than enprostil, which was greater
than PGD2. These results indicate the existence of EP1,
EP2, and EP3 as well as IP subtype receptors on human monocytes.
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Influence of Age and Sex on Prostaglandin Binding
to Monocytes
To study the influence of age on the binding of 3H-PGs
to human monocytes, the binding data of the controls were
analyzed for two groups with mean ages of 27±6 years and
56±11 years. We found no significant difference between these two age
groups with regard to the binding of 3H-PGs (Tables 1, 4,
6). Similarly, the binding data derived from the patients were
analyzed for female and male patients separately; no difference
was found between the sexes (Tables 2, 5, 7).
CAMP Formation
Prostaglandins dose-dependently enhanced cAMP formation by
monocytes in both healthy control subjects and patients. The most
potent prostaglandin was PGE2 with a
concentration that caused elevation by 50% of basal cAMP
(ED50) of 3.8±2.4 nmol/L in patients and 1.6±1.2
nmol/L in healthy control subjects (P<.01).
PGI2 was about two times less effective, with an
ED50 of 5.6±4.1 nmol/L in patients and 3.1±1.4
nmol/L in healthy control subjects. PGE1 was also a
weaker agonist than PGE2, with an ED50 of
6.3±3.5 nmol/L in patients and 4.8±2.5 nmol/L in
healthy volunteers (Table 8).
Effect of Lipoproteins on Prostaglandin Binding to
Monocytes
Treatment with acetylated LDL or oxidized LDL resulted in
a significant (P<.01) decrease in prostaglandin
binding sites to monocytes both in patients and control subjects
(Tables 1, 4 through 7). Native LDL and HDL did not significantly
affect prostaglandin binding. The effect of modified
LDL on prostaglandin binding to monocytes was slightly more
pronounced in patients, however, without statistical significance
between both groups.
Effects of Lipoproteins on cAMP Formation by Monocytes
Incubation of monocytes with modified LDL (acetylated LDL
and oxidized LDL) led to a significant decrease in
prostaglandin-stimulated cAMP formation of monocytes
derived from control subjects as well as from patients
(P<.05-P<.01), whereas native LDL and HDL
showed no significant effect on prostaglandin-stimulated
cAMP-production (Table 8).
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Discussion |
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In addition to the classic LDL receptor, monocytes and macrophages also express scavenger receptors; one class of receptors has a higher affinity for acetylated LDL, another prefers oxidized LDL, and a third one binds both modified LDL molecules.29 Modified LDLs have been shown to interfere with the atherogenic process in different ways, such as leading to endothelial cell injury, stimulating monocytes into the arterial wall, inhibiting the movement of tissue monocytes, and generating foam cells by uptake of modified LDL via the scavenger receptors.
PGE1, PGE2, and PGI2 are considered to exert protective effects against atherosclerosis. They are potent inhibitors of platelet aggregation and vasoconstriction as well as inhibitors of monocyte macrophage activation.30 The results of this study indicate the existence of saturable and specific binding sites for PGE2 and PGI2 as well as PGE1 on human monocytes.
A classification of prostanoid receptors into DP, EP, FP, IP, and TP
for each of the five naturally occurring prostanoids, PGD2,
PGE2, PGF2, PGI2,
and TXA2 was recently proposed by Coleman et
al.27 Furthermore, four subtypes of EP receptors, namely
EP1, EP2, EP3, and EP4 have been described.27 The
existence of a PGE2 receptor on monocytes and
macrophages was suggested previously.12 13 15 In
the present study, 3H-PGI2 binding to
monocytes was best displaced by cicaprost (a selective IP receptor
agonist), PGI2, and iloprost, which suggests the existence
of the IP receptor on human monocytes. At the
3H-PGE2 receptor level, the best competitors
were PGE2, PGE1, enprostil (a selective EP3
receptor agonist), followed by 17-phenyl-PGE2, (a relative
selective EP1 receptor agonist), iloprost, PGI2 and
butaprost (a selective EP2 receptor agonist). At the
3H-PGE1 receptor level, the best competitors
were PGE1, 17-phenyl-PGE2, enprostil, iloprost
and PGE2; PGD2 and butaprost were weaker
agonists. These findings also suggest the existence of EP1,
EP2, and EP3 subclasses of the PGE receptor on
human monocytes.
Recent evidence has shown the heterogeneity of PGE1-binding sites for other cell types as well.31 One subtype of the PGE1 receptor binds PGI2, the other one binds PGE2. As with other leukocytes,8 the binding of 3H-PGE1 to the monocyte PGE1 receptor could be displaced by iloprost (a PGI2-analogue with substantial affinity for PGE1 receptors) as well as by PGI2.27 Also the binding of 3H-PGI2 to the PGI2 receptor could be displaced by unlabeled PGE1. This competition behavior suggests the existence of a PGE1-PGI2 binding protein on the cell surface of human monocytes. Whether this monocyte receptor protein differs from those expressed on other cells such as basophils8 or platelets10 remains to be investigated.
On the cell surface of monocytes obtained from patients with peripheral vascular disease and hyperlipoproteinemia IIa we found a significantly lower capacity and affinity of binding sites for each of the radiolabeled prostaglandins investigated (PGE1, PGI2, and PGE2) compared with the normolipemic volunteers. This significant difference is shown not to be influenced by age or/and sex (Tables 1, 4 through 7). One possible explanation for the difference in the number of prostaglandin-binding sites may be the interference with certain lipoproteins and/or with endogenously produced ligands. In fact, recent investigations suggest that certain lipoproteins can stimulate AA-metabolism of monocytes and macrophages leading to prostaglandin and leukotriene formation.32 33 34 35 Hartung et al32 found that stimulation of the scavenger receptor by acetylated LDL promotes the generation of eicosanoids by human monocyte-macrophages. However, metabolism of AA in mouse peritoneal macrophages was affected only by oxidized LDL, while ß-VLDL, native LDL, and acetylated LDL had no stimulatory effect.33 Diez and colleagues34 reported that acetylated LDL promotes the release and metabolism of AA by murine macrophages, while native LDL maintains no effect. By contrast, Jambou et al35 reported that although native LDL stimulates mainly thromboxane and PGI2 formation in the mouse macrophage cell line P388D1, HDL stimulates mainly the production of PGI2. In addition, Habenicht et al36 reported that LDL stimulates prostaglandin production in platelet-derived growth factor –stimulated cells by providing substrate-AA for the PGH-synthase, the rate-limiting enzyme of PG synthesis. This effect was dependent on the LDL receptor pathway suggesting that prostaglandin formation is directly linked to the LDL pathway in monocytes and macrophages. These inconsistent results may, in part, be explained by the use of cells of different origin or different experiment conditions.
In this study we also investigated the binding capacities and
affinities for the three prostaglandins after incubation of
monocytes with different lipoproteins. Tables 1, 4 through 7 indicate
that significantly fewer PGE1-, PGE2-, and
PGI2-binding sites were expressed on monocytes isolated
from healthy control subjects as well as patients after incubation with
modified LDL; however, native LDL and HDL did not alter the
prostaglandin-binding behavior. Interestingly, decreased
Bmax values (binding density) and unchanged Kd
values (binding affinity) were found after addition of modified LDL.
The shape of the binding isotherms allow the exclusion of conditions
that would normally lead to an increase in Kd values, such
as competition of prostanoids or prostaglandin-like
oxidation products in the modified LDL particle for the binding of
labeled prostaglandin; interaction of labeled
prostaglandin with LDL in the solution, which would lead to
a decrease in the total available prostaglandin
concentration; or competition of endogenous
prostaglandin due to stimulation by modified LDL. The last
condition appears impossible because in our studies the
endogenous prostaglandin synthesis of monocytes
was inhibited by indomethacin. It was demonstrated that
indomethacin suppressed the generation of PGE,
TXB2, and 6-keto-PGF1 of
macrophages induced by acetylated or
malondialdehyde-modified LDL.32 Therefore, the effects
observed in this study, ie, reduced prostaglandin-binding
properties and altered cAMP concentration might not only be due to
production of prostaglandin induced by modified
LDL. It is likely that the decrease in Bmax values reflects
a downregulation or upregulation of binding proteins. As shown in this
study, downregulation of receptors is induced by modified LDL but not
by native LDL. The feedback mechanism to PGE receptor density suggests
a regulatory link between modified LDL and the expression of
prostaglandin receptors on human monocytes. It remains to
be investigated whether this feedback mechanism is formulated via the
scavenger receptor or whether modified LDL induces other AA
products of the lipoxygenase pathway (or both). The
latter seems possible, because, as demonstrated by Fair et
al,37 oxidized LDL increases mononuclear cell
production of 5-hydroxyeicosatetraenoic acid and
15-hydroxyeicosatetraenoic acid. It has been suggested that major
changes occur in the surface organization of lipoproteins in response
to PGE1.38 Manevich et al38 found
that low concentrations (10-13 to
10-9 mol/L) of PGE1 and
PGF2 induced rearrangement of the LDL
surface, whereas PGE2 had no effect. In the present
study we used about 2 nmol/L of LDL, a concentration which
corresponds to the half-maximal saturation dose for PGE1
binding. Assuming an interaction of PGE1 with LDL, this
would lead to a decrease in the total PGE1 concentration
available in the reaction mixture, and this decrease would amount, in
the presence of 2 nmol/L PGE1, to 0.16% (ie, 600
particles of LDL bind two particles of PGE1). Because of
this low binding capacity of LDL for PGE1, the total
concentration of PGE1 will be lowered by approximately
0.16%. Moreover, if such an interaction between
prostaglandin and lipoprotein exists, it should result in
decreasing Kd values, with Bmax values
remaining unchanged (if such "complexes" would lead to
decreased concentrations of unbound prostaglandins). Since
native LDL does not show an influence on
prostaglandin-binding parameters it seems
unlikely that changes in the prostaglandin receptor density
and/or affinity in the presence of modified LDL has any relation with
an interaction between modified LDL and prostaglandin in
solution. Of course, another possibility would be that the interaction
of PGE1 with LDL changes the LDL particle so much that it
causes the decrease in Bmax level. This, however, is
unknown. In any case, a lower PGE receptor density means a lower
sensitivity for prostaglandins.
In agreement with the fewer receptors found for monocytes isolated from
patients cAMP levels were also decreased. cAMP is considered to be the
second messenger for mediating the actions of
prostaglandins; it has been shown to stimulate efflux of
intracellular sterol from cholesterol-loaded cells such as
endothelial cells and fibroblasts.39 In
this study we observed a significantly lower level of both basal as
well as prostaglandin-stimulated cAMP in patients with
peripheral vascular disease compared with normolipemic
subjects. Furthermore, as shown in Table 8, incubation of monocytes
with modified LDL resulted in increased ED50 values, ie,
the capacity of prostaglandin-stimulated cAMP formation in
monocytes was reduced, which corresponds with the fewer receptors
estimated after preincubation with modified LDL. This finding suggests
that fewer prostaglandin receptors and/or lower affinity of
receptors to prostaglandins are associated with a decreased
adenylate cyclase activity in monocytes.
This study shows that decreased PGE1, PGE2, and PGI2 receptor/adenylate cyclase activity of monocytes in hypercholesterolemic patients may be regulated by modified LDL. These observations may have further important implications for the understanding of atherogenesis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the "Jubiläumsfonds" of the Austrian National Bank (Nos. 4560 and 5439).
Received July 7, 1995; accepted March 1, 1997.
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References |
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