© 1998 American Heart Association, Inc.
Original Contributions |
Atherogenic Properties of Enzymatically Degraded LDL
Selective Induction of MCP-1 and Cytotoxic Effects on Human Macrophages
From the Institute of Medical Microbiology and Hygiene, Johannes-Gutenberg University of Mainz, Mainz, Germany.
Correspondence to Dr Mariam Klouche, Johannes-Gutenberg University of Mainz, Institute of Medical Microbiology, Obere Zahlbacher Strasse, 55101 Mainz, Germany. E-mail klouche{at}mail.Uni-Mainz.de
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Abstract |
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Abstract—The mechanisms underlying the selective accumulation of macrophages in early atherosclerotic lesions are poorly understood but are likely to be related to specific properties of altered low density lipoprotein (LDL) deposited in the subendothelium. Enzymatic, nonoxidative degradation of LDL converts the lipoprotein to a potentially atherogenic moiety, enzymatically altered LDL (E-LDL), which activates complement and is rapidly taken up by human macrophages via a scavenger receptor–dependent pathway. Immunohistological evidence indicates that E-LDL is present in an extracellular location in the early lesion. We report that E-LDL causes massive release of monocyte chemotactic protein 1 (MCP-1) from macrophages and that expression of interleukin 8 or RANTES remains unchanged. Release of MCP-1 was preceded by a rapid expression of MCP-1 mRNA, which was detectable after 15 minutes, reached maximum levels after 1 hour, and remained detectable for 12 hours after exposure to concentrations as low as 10 µg/mL E-LDL. MCP-1 mRNA induction and protein release by E-LDL exceeded that evoked by oxidized LDL. Release of MCP-1 was dependent on de novo protein synthesis and on the activity of tyrosine kinases. At higher concentrations, E-LDL, but not oxidized LDL, exerted toxic effects on macrophages that in part appeared to be due to apoptosis. The results show that E-LDL possesses major properties of an atherogenic lipoprotein.
Key Words: atherosclerosis • macrophages • LDL • monocyte chemotactic protein 1 • toxicity
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Introduction |
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Selective recruitment of monocytes and macrophages into the artery wall is one of the earliest events in the pathogenesis of atherosclerosis. Because subendothelial lipid deposition is accompanied by complement activation,1 2 3 one explanation for the initial attraction of blood leukocytes could be the local generation of chemotactic complement products. However, complement anaphylatoxins exhibit no preference for monocytes. Their selective recruitment during lesion progression thus remains an enigma that might be resolved if a component of the fatty streak was found to induce the production of a specific monocyte attractant, such as monocyte chemotactic protein 1 (MCP-1).4 5 In vivo expression of MCP-1 has been detected in human and rabbit atherosclerotic lesions, particularly within macrophage-rich regions, and to a lesser extent in areas with predominant smooth muscle cells.6 7 Although all cell types present in the atherosclerotic lesion have the potential to secrete MCP-1,7 8 9 10 11 subendothelial macrophages appear to be the predominant source of MCP-1 throughout the different stages of atheroma.12 Additionally, endothelial and smooth muscle cells can stain positively for MCP-1 in early and advanced lesions, respectively.12 In cholesterol-fed rabbits, expression of MCP-1 mRNA by macrophages is confined to cells in the atherosclerotic lesions, whereas macrophages from normal vessels or alveolar macrophages of the same animals do not express MCP-1.6 This indicates that a lesion component with MCP-1–inducing properties exists. In this context, it has been shown that smooth muscle and endothelial cells express MCP-1 in vitro after treatment with minimally modified low density lipoprotein (LDL),8 9 a finding that is in line with the widely held belief that oxidized LDL (ox-LDL) is the major atherogenic lipid.
We previously reported that enzymatic degradation of LDL also generates a potentially atherogenic molecule. Enzymatically altered LDL (E-LDL) displays the same micromorphological characteristics as lipid droplets that were visualized in and isolated from atherosclerotic lesions.13 E-LDL, but not ox-LDL, activates complement, is recognized by a macrophage scavenger receptor, and is a potent inducer of foam cell formation. Recently, immunohistochemical data obtained through the use of specific monoclonal antibodies indicated that E-LDL is extensively deposited extracellularly in the early atherosclerotic lesion.14
The question thus arises of whether E-LDL might induce expression and release of chemotactic factors or exert cytotoxic effects. The results reported here corroborate the concept that E-LDL may be a relevant factor in atherogenesis.
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Methods |
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Isolation and Culture of Human Monocyte-Derived Macrophages
Human monocytes were obtained from buffy coats of 45 healthy blood donors, courtesy of the blood bank of the University of Mainz, by density centrifugation.13 Purity was >90%, with lymphocytes as major contaminating cells, as determined by flow cytometry. Viability was >95%, as determined by trypan blue exclusion. Plating efficiency was 97% to 100%. Monocytes adjusted to 106 cells/mL were maintained in minimal essential medium supplemented with 10% human nonlipemic AB serum, penicillin (100 U/mL, Gibco-BRL), streptomycin (100 µg/mL, Gibco-BRL), interferon gamma (1 ng/mL, Bioferon), and granulocyte macrophage–colony-stimulating factor (2.5 ng/mL, Sandoz/Essex) to promote monocyte differentiation. Adherent macrophages were cultivated at 37°C in 5% CO2, and medium was changed every 3 days. For experiments, macrophages were used on days 6 to 10 of culture and maintained in the absence of serum.
Lipoprotein Isolation
Plasma used for the isolation of LDL was obtained from healthy
blood donors, aged 18 to 65 years, who had no diabetes or hypertension,
did not take any medication, and had no signs of ischemic heart
disease. Native LDL (d=1.020 to 1.062 g/mL) was isolated by
a modified method based on preparative sequential
ultracentrifugation in KBr gradients. After isolation,
LDL was dialyzed against Tris-NaCl buffer (5 mmol/L Tris and
150 mmol/L NaCl, pH 7.4) containing 1 mmol/L EDTA to prevent
artificial oxidation. Cholesterol content, determined by an
enzymatic test (Boehringer-Mannheim), ranged from 5 to 9.5
mg/mL, and protein content, analyzed by the Bradford method
(Roth), ranged from 3.6 to 7 mg/mL.
Modification of LDL
Chemical and enzymatic modifications of native LDL were
conducted as described by Bhakdi et al.13 In
brief, LDL was subjected to 3 enzymatic treatments consisting of
trypsin (6.6 µg/mL, Sigma), cholesterol esterase (40
µg/mL, Boehringer-Mannheim), and neuraminidase (79 mU,
Behring).13 Treatment with trypsin and
cholesterol esterase was conducted at 37°C for 2 hours
(pH 7.4), followed by a 10-hour treatment with neuraminidase. To
provide optimal conditions for the action of neuraminidase, pH was
lowered to 4.9 by addition of 50 mmol/L MES, which terminated the
action of trypsin and cholesterol esterase. Extensive
oxidative modification of LDL was performed according to published
protocols using 50 µmol/L
CuSO4.15 Before
modification, LDL was adjusted to a concentration of 3 mg/mL
cholesterol. The modified LDL preparations were stored at
4°C and used within 2 weeks. All lipoprotein concentrations are
expressed in micrograms of cholesterol per milliliter.
During LDL modifications, general precautions were taken to avoid
lipopolysaccharide (LPS) contamination, which was further
excluded in control assays yielding negative results with the
Limulus endotoxin assay (E-Toxate, Sigma).
Assays for Products of Lipid Peroxidation
Thiobarbituric acid–reactive substances were measured as
described previously.16 E-LDL contained no
detectable amounts of these substances, and ox-LDL contained mean
amounts of 70 to 80 nmol/mg cholesterol.
Reverse Transcription–Polymerase Chain Reaction (RT-PCR) of
Chemokine and Cytokine mRNA
Total cellular RNA was extracted from human monocyte-derived
macrophages by guanidine isothiocyanate-phenol-chloroform
extraction as described by Chomczynski and
Sacchi.17 Lysis was conducted directly on culture
plates, and 6 to 12x106 macrophages were
used for extraction of RNA per analysis. Initially, the mRNA in
aqueous solution was denatured at 65°C for 5 minutes and then chilled
on ice. Reverse transcription of 1 µg of total RNA was performed
using avian myoblastosis virus reverse transcriptase for 4 hours at
37°C with standard reagents (Promega). For polymerase chain reaction,
2 µL of the cDNA product (40 ng) was amplified in a 50-µL
reaction volume with the specific primer pairs and Taq
polymerase in a modified protocol provided by the manufacturer
(Gibco-BRL). PCR was conducted at 95°C for an initial denaturation of
5 minutes, followed by 20 cycles of denaturation at 95°C for 40
seconds, annealing at 62°C for 1 minute, and extension at 72°C for
3 minutes. Final extension was 10 minutes at 72°C (Biometra cycler).
Samples lacking cDNA or RNA served as negative controls. The PCR
products were run on 1% agarose gels in 1x Tris-borate-EDTA and
stained with ethidium bromide unless indicated otherwise.
Intron-spanning sets of primers were selected, and sequencing of
individual clones confirmed that the amplified fragments were identical
with the published human sequences. Sequencing was performed on cloned
amplification products by the dideoxy chain termination
method18 using the dideoxy terminator cycle
sequencing kit (PE Applied Biosystems). Sequencing products were
separated and analyzed using a 373A automated sequencer (PE
Applied Biosystems). The
Table
lists the primer
sequences used and the length of each predicted product.
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Quantification of MCP-1 Gene Expression by Competitive
RT-PCR
Expression of specific MCP-1 gene transcripts was quantified by
competitive RT-PCR using a synthetic DNA as an internal
standard.19 For quantitative analysis,
target MCP-1 mRNA and the internal standard were coamplified in 1
reaction tube using the same primers. The amplification products
could then readily be separated by gel electrophoresis because the
internal standard yielded a PCR product of a different size. By
using serial dilutions of the internal standard, the amount of target
cDNA could be quantified by extrapolating against the standard
concentration after scanning densitometric analysis.
The internal standard was produced by cloning the MCP-1 amplification product into a TA vector (pCR3, Promega). A random DNA fragment (<200 bp), obtained by AluI digestion of pUC19, was subcloned into the unique NruI restriction site of the MCP-1 sequence so that the plasmid contained MCP-1 exons 1 to 3 and a 120-bp insertion between exons 1 and 2, as confirmed by sequencing. Restriction enzymes and T4 ligase were obtained from Boehringer-Mannheim. Both competitor and cDNA showed identical amplification efficiency, with optimal products at 20 PCR cycles. All standard procedures were performed as recommended by Sambrook et al.20
Digital Image Analysis
Quantitative image analysis of the RT-PCR products
was performed with a scanning densitometer coupled to a computer-based
densitometric measurement imaging system (Herolab). The recorded
intensities of the ethidium bromide fluorescence signals were
determined for each lane as the sum of pixel values [uncalibrated
values, (picture points)2 xgray values]. In the
exponential phase of amplification, the amount of target cDNA could be
derived by comparing bands generating equal intensities with the known
amount of standard.
Chemokine and Cytokine Assays
The amounts of chemokines and cytokines released in the
supernatant of lipid-treated macrophages were determined by
enzyme immunoassay. Sources for the commercial kits were as follows:
MCP-1 and RANTES (regulated upon activation, normal T-cell expressed
and presumably secreted), R&D Biosystems; interleukin (IL) 8,
Innogenetics; and IL-1 and IL-6, Medgenix.
Chemotaxis Assay
The biological activity of MCP-1 released by E-LDL–stimulated
macrophages was determined in 48-well microchemotaxis chambers
(Nucleopore). Macrophage supernatants were assayed in dilutions
from 1:1 up to 1:500 to account for the narrow range of the chemotactic
activity of chemokines. In addition, supernatant dilutions were
incubated with anti–MCP-1 antibody (1:200) (Pharmingen).
Macrophage supernatants with or without anti–MCP-1 antibodies
were placed in the lower wells and separated by polycarbonate filters
(pore size, 5 µm) from the upper wells, which contained
105 human monocytes. As a control,
isotype-matched antibodies against an irrelevant antigen were used;
these antibodies did not affect macrophage chemotaxis.
Chemotaxis was conducted for 90 minutes at 37°C in a humidified
atmosphere. The numbers of migrating monocytes was determined in 5
high-power fields. The chemotactic index was calculated by dividing the
mean number of monocytes that migrated into macrophage
supernatants by the number of migrated monocytes in medium alone
(spontaneous, nondirected migration) ±SD.
Detection of Cellular Toxicity
Cytotoxic effects mediated by E-LDL were evaluated by
quantifying the reduction of intracellular ATP and the release of
lactate dehydrogenase into the supernatant. For the determination of
cellular ATP levels, cells were lysed by the addition of 1% Triton
X-100 (200 µL). The lysates were immediately mixed with a suitable
dilution of ATP-luminol reagent (Boehringer-Mannheim), and
luminescence was determined in a Biolumat LB 9500 (Berthold). Dilution
of the luminol reagent for each individual experiment was determined to
obtain ATP measurements within the linear range. LDH was measured by
mixing macrophage supernatants with triethanolamine (250
mmol/L, pH 7.6), EDTA (125 mmol/L, pH 7.0), and NADH in TRIZMA
buffer (50 mmol/L, pH 7.5). After the addition of pyruvate
(Seromed), absorbance was determined at 340 nm. Results for ATP and LDH
concentrations were expressed as a percentage of values obtained with
control cells that had been incubated with the enzyme mix without
E-LDL.
Detection of DNA Strand Breaks
The terminal
deoxynucleotidyltransferase
(TdT)–mediated dUTP nick-end labeling (TUNEL) method was used to
detect single- and double-stranded DNA breaks that occur at early
stages in apoptosis. The TUNEL staining kit was obtained from
Boehringer Macrophages grown on chamber slides (Nunc)
were fixed and stained following the manufacturer's instructions.
Control cells were exposed to the enzyme mix alone. Specimens were
visualized by fluorescence microscopy, and the number of
TUNEL-positive cells was estimated by counting.
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Results |
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E-LDL Induces Expression and Release of MCP-1 From Human Macrophages
Treatment of human macrophages with E-LDL resulted in very rapid induction of MCP-1 mRNA. MCP-1 expression began 15 minutes after exposure to 100 µg/mL E-LDL (Figure 1A
). Peak MCP-1 mRNA levels were observed
after 1 hour of challenge, and MCP-1 expression was maintained for >12
hours. The expression of the housekeeping gene GAPDH
(glyceraldehyde-phosphate dehydrogenase) was
analyzed as a control. The release of MCP-1 protein lagged
behind the peak of mRNA expression and increased over 8 hours, showing
maximum levels after 24 hours of stimulation (Figure 1B). As in all the
following experiments, controls incubated with equivalent amounts of
the enzyme mixture remained negative.
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As opposed to the rapid kinetics of MCP-1 induction, the constitutive
expression of IL-8 by macrophages remained unaltered in the
presence of E-LDL. E-LDL also exerted no effect on the expression of
RANTES (data not shown). However, E-LDL did induce some expression of
IL-6 and IL-1 mRNA. In contrast to the expression of MCP-1, expression
of mRNAs of the inflammatory cytokines showed only a short peak
between 30 minutes and 6 hours of incubation (Figure 2A). Only minor amounts of IL-6 protein
were liberated after 12 hours of stimulation with E-LDL, reaching
maximal levels of 0.15 ng/mL, whereas no augmented release of IL-1
protein was measurable (Figure 2B).
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Quantification of MCP-1 Expression by Competitive RT-PCR
To quantify MCP-1 mRNA, an internal standard was constructed that
contained identical primer binding sites but differed in size from the
original cDNA. The cDNA was coamplified with varying amounts of the
internal standard. The results of an experiment conducted after a
2-hour incubation of human macrophages with 100 µg/mL E-LDL
are shown in Figure 3. Initially, the
cDNA was coamplified with serial 10-fold dilutions of the internal
standard with the corresponding densities given below (Figure 3A).
Equimolar amounts of the internal standard and the cDNA were observed
after further 2-fold dilutions of the competitor DNA,
representing 0.35x10-12 mol/L
(Figure 3B). The continuous presence of E-LDL was not required for the
expression or release of MCP-1, because both intermittent and
continuous treatment with the modified lipoprotein resulted in almost
identical levels of mRNA (data not shown).
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E-LDL Is a More Potent Inducer of MCP-1 Expression Than
ox-LDL
Figure 4 shows the induction
kinetics of MCP-1 mRNA expression after treatment of
macrophages with E-LDL, ox-LDL, native LDL (each at 100 µg/mL
cholesterol), or LPS (1 µg/mL). Unstimulated human
macrophages did not constitutively express detectable amounts
of MCP-1 mRNA. After only 15 minutes of exposure to 100 µg/mL E-LDL,
a 17-fold induction of MCP-1 expression was observed. Maximum
expression occurred after 1 hour of exposure, corresponding to an
increase by a factor of 250. Ox-LDL also induced MCP-1 mRNA, but with
reduced potency. Maximal expression was also observed after 1 hour of
incubation, with peak levels less than one third of those observed with
E-LDL. Native LDL provoked very little MCP-1 mRNA synthesis; this was
observed in all experiments in which E-LDL and LDL were always derived
from identical batches. E-LDL was almost as effective as LPS (1
µg/mL), but induction was more rapid, with the mRNA peak preceding
that after LPS stimulation by almost 3 hours. In view of these markedly
differing kinetics, contamination of the lipoprotein preparations with
LPS appeared unlikely. To further exclude this, incubations were
performed in the presence of 10 µg/mL polymyxin B, which completely
blocked the effect of LPS (1 µg/mL) on MCP-1 induction but did not
inhibit the effects of E-LDL.
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Dose-Dependent Release of MCP-1 From Macrophages Induced
by E-LDL
E-LDL stimulation of macrophages resulted in a
dose-dependent induction of MCP-1 mRNA, commencing in the presence of
10 µg/mL E-LDL (Figure 5), with a
maximum at 100 µg/mL E-LDL and a sharp decline thereafter.
Quantitative analysis showed that 
16 000 molecules of MCP-1
mRNA per nanogram of total RNA were present at maximal induction
(Figure 5, filled circles). If the protein synthesis
inhibitor cycloheximide (10 µg/mL) was added during
transcription, MCP-1 mRNA did not accumulate. This was the case even
when cycloheximide was added 1 hour after the transcription had been
optimally induced by E-LDL. These results indicate that E-LDL does not
induce the concomitant expression of RNAses.
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,
right scale) are shown in the middle panel. Induction of MCP-1 mRNA
(bottom) is accompanied by dose-dependent MCP-1 protein release (
)
after stimulation with E-LDL. In contrast, no effect was observed on
the release of IL-8 (
). Incubation time was 12 hours. Points
represent mean values (±SD) of 6 experiments.
MCP-1 mRNA expression was accompanied by a dose-dependent MCP-1 protein
release (Figure 5
, filled squares). In the presence of only 10 µg/mL
E-LDL, the release of MCP-1 increased nearly 6-fold over baseline
liberation. Maximum release occurred at 100 µg/mL E-LDL, resulting in
the secretion of >8 ng/mL MCP-1. During autumn and winter, an
increased number of blood donors had leukocytosis (10 000 to
12 000/µL) or elevated acute-phase proteins indicative of concurrent
infections. The monocyte-derived macrophages of these donors
spontaneously released up to 1 ng/mL MCP-1 into the supernatants and
expressed MCP-1 mRNA, but at an 150-fold reduced level compared with
optimal stimulation with E-LDL. The protein data presented
include these spontaneous MCP-1 producers, which were retrospectively
excluded from the RT-PCR analyses. Human monocyte-derived
macrophages constitutively secreted IL-8, which could be
detected at concentrations of up to 1.3 ng/mL in unstimulated
supernatants. In contrast to MCP-1, no enhancement of this IL-8 release
was observed at any concentration of E-LDL applied (Figure 5, unfilled
squares). Similarly, RANTES was not liberated by E-LDL stimulation
(data not shown). Cycloheximide and the protein tyrosine kinase
inhibitors herbimycin A and genistein completely inhibited
E-LDL–induced release of MCP-1 (Figure 6).
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MCP-1 Released by E-LDL–Treated Macrophages is
Biologically Active
Macrophage supernatants recovered after 12 hours of E-LDL
treatment were assayed for chemotactic activity. Optimal monocyte
attraction, indicated by a chemotactic index of 2.7, was observed at a
supernatant dilution of 1:100, which corresponded to an MCP-1
concentration of 10 nmol/L. That the chemotactic activity was
attributable to MCP-1 was confirmed by the addition of a neutralizing
anti-human MCP-1 antibody, which reduced monocyte chemotaxis by 56%
(Figure 7). No reduction was observed
when an isotype-matched, irrelevant monoclonal antibody was used.
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), which was reduced in the presence of
the specific anti-human MCP-1 antibodies (aMCP-1, 
). The chemotactic
index was calculated as the number of monocytes counted after
stimulated migration divided by the number of monocytes that migrated
spontaneously, as determined in 9 high-power fields. Points
represent mean values (±SD) of 3 experiments.
Toxicity of E-LDL to Human Macrophages
At neutral pH, E-LDL concentrations of up to 100 µg/mL were not
cytotoxic to human macrophages, even when present for >24
hours. Under these conditions, macrophages rapidly take up
E-LDL.13 However, at concentrations >200
µg/mL, E-LDL exerted toxic effects, as shown by a gradual loss of
intracellular ATP and release of LDH (Figure 8). A reduction in ATP of 50%
occurred after 12 hours of incubation with 400 µg/mL E-LDL.
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). Toxicity was expressed as the
percentage of ATP or LDH of untreated controls. Points
represent mean values (±SD) of 6 experiments.
When the pH of the medium was slightly lowered to 6.5, the cytotoxic
effects were accentuated. Figure 9 shows
results of an experiment conducted at neutral and slightly acidic pH.
In the latter case, ATP depletion started at E-LDL concentrations of 50
µg/mL and massive cell death was observed at 400 µg/mL E-LDL. At pH
6.5, the bell-shaped MCP-1 release curve (Figure 5) also shifted to the
left and reached a maximum at E-LDL concentrations of 50 µg/mL
(data not shown).
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Figure 10 shows the results of TUNEL
staining of macrophages after incubation with 400 µg/mL E-LDL
for 8 hours at pH 7. The number of positively staining cells in
controls was estimated to be <2% in all experiments, whereas positive
staining was always observed after incubation with 200 to 400 µg/mL
E-LDL (n=5). By visual enumeration, it was estimated that 10% to 15%
of the cells stained positively after 8 hours of incubation with 400
µg/mL E-LDL, pH 7.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Atherosclerosis progresses from a precursor lesion characterized by subendothelial insudation of LDL to a complicated lesion with predominant tissue necrosis and fibrosis. Between these extremes, the lesion shows classic features of a chronic inflammatory process. Current interest is focused on understanding the pathogenesis of the early lesion. Unanswered questions relate to the nature of the LDL modification that renders the molecule atherogenic and to mechanisms by which inflammation is upheld. That LDL needs to be modified to be efficiently taken up by macrophages is known.21 22 Besides the currently favored concept of oxidative modification of LDL, a number of other modifications convert LDL to forms that are taken up by macrophages via scavenger receptors or Fc-receptor pathways. Witztum and Steinberg23 have conceded that modifications other than oxidation may play an additional, if not dominant, role in the development of atherosclerosis.
In a previous study, we discovered that enzymatic degradation renders LDL potentially atherogenic.13 E-LDL is avidly taken up by human macrophages. In contrast to ox-LDL, E-LDL is capable of activating complement. That E-LDL may truly be relevant in vivo is supported by the recent finding that degraded LDL is present in the early atherosclerotic lesion, as demonstrated by immunohistochemical tests.14
Now, we show that E-LDL is endowed with a remarkable potential to induce MCP-1 release from human macrophages. These results could satisfactorily account for a number of key findings in the literature. In vivo expression of MCP-1 mRNA and protein has been detected primarily in macrophage-rich areas of atherosclerotic lesions in humans and animals.6 7 11 12 24 25 Consistent with its presumed pathophysiological role in atherosclerosis, expression of MCP-1 mRNA and protein coincide with the neointimal infiltration of macrophages.26 Moreover, from experimental models of atherosclerosis, it is evident that expression of MCP-1 is confined to lesional macrophages.6 This strongly suggests the presence of a specific lesion component with MCP-1–stimulating activity.6 MCP-1 induction by E-LDL exhibits remarkably rapid kinetics; similarly rapid RNA induction peaking at 1 hour was previously described for acetylated LDL.27
The MCP-1–inducing potency of E-LDL markedly exceeds that of ox-LDL. We found E-LDL to be at least 3 times more potent than ox-LDL in inducing MCP-1 mRNA in human macrophages. By Northern blot analysis, an up to 22-fold increase of MCP-1 mRNA was recently estimated to be induced by minimally modified (oxidized) LDL in human endothelial and smooth muscle cells.9 This magnitude of induction is comparable with our results obtained after stimulation of macrophages with ox-LDL. The inhibitory effects of the protein tyrosine kinase inhibitors genistein and herbimycin28 29 indicate that that the activation of mitogen-activated protein kinases30 is required for the action of E-LDL.
Ox-LDL has been reported to induce IL-8
secretion31 and expression of IL-8
mRNA27 in the monocytic cell line THP-1, but we
found no enhancement of IL-8 expression in human macrophages by
E-LDL. We have not investigated whether this might have been due to our
use of monocyte-derived macrophages rather than the THP-1 cell
line. The selective induction of MCP-1 appears to be a specific
property of E-LDL, because simple phagocytosis of zymosan-particles
stimulates the concomitant release of IL-8, tumor necrosis factor-
,
and MCP-1 (data not shown). Neither ox-LDL nor E-LDL induced RANTES in
our experiments. Failure of E-LDL to promote IL-8 secretion would be in
line with the paucity of granulocytes in progressing lesions.
E-LDL also transiently stimulated the expression of IL-1 in human macrophages, which could be in line with in vivo results showing expression of IL-1 in human abdominal aneurysms.32 However, no enhanced secretion of IL-1 could be discerned in our studies. We observed slight stimulation of IL-6 mRNA and protein release by E-LDL. The amounts of IL-6 released in response to E-LDL were low, and the biological relevance remains to be assessed.
E-LDL also exerted cytotoxic effects on macrophages. At neutral
pH, the toxic effects were observed at E-LDL concentrations 
200
µg/mL. Plasma LDL levels are well above 1000 µg
cholesterol/mL, so the cytotoxic doses of E-LDL determined
here are thought to be realistic. The cytotoxic effects were markedly
accentuated at pH 6.5. Because it is conceivable that the local
extracellular pH in areas of chronic inflammation is lowered, the
present finding may turn out to be relevant. Direct toxic effects
have also been demonstrated for ox-LDL.23 33
Positive TUNEL stainings were always observed in a fraction of foam
cells, consistent with apoptotic cell death.
Preliminary estimates ranged from 10% to 15% for cells treated with
400 µg/mL E-LDL at pH 7 after 8 hours. Relative ATP depletion was
50% at this stage, possibly because ATP reduction can have multiple
causes, including increased consumption. Indeed, initial ATP
consumption may be 1 factor contributing to the events that culminate
in DNA degradation. The mechanisms underlying E-LDL cytotoxicity remain
to be elucidated. However, our findings are in good accord with recent
immunohistological data revealing the presence of
apoptotic macrophages and smooth muscle cells in
advanced human atherosclerotic lesions.34 35 36 In
vitro, ox-LDL induces apoptosis in cultured human smooth muscle
cells, apparently mediated by the ketosterol and hydroxysterol
fractions, respectively.37 38
A straightforward concept based on the collective data is that transformation of subendothelially deposited LDL to an atherogenic moiety can occur simply by enzymatic, nonoxidative degradation. Treatment of LDL with a variety of proteases39 40 or with a protease plus cholesterol esterase41 has been found by other investigators to increase its atherogenicity. Evidence of the presence of cholesterol esterase in the subendothelium is available,42 and our immunohistochemical data directly demonstrate that subendothelially deposited, extracellular LDL is enzymatically modified in the early lesion.14 In vitro, the additional action of neuraminidase enhances the atherogenic potential of LDL. It is of interest that the amount of sialic acid in LDL has been reported to correlate inversely with atherosclerosis.43 44 Thus, we hypothesize that maximal atherogenicity is bestowed onto LDL through enzymatic degradation with all 3 enzymes. This enzyme treatment causes LDL fusion with the formation of heterogeneously sized lipoprotein particles. It remains to be established whether the chemokine-inducing and cytotoxic properties are preferentially expressed by certain subfractions. However, it is important to note that Frank and Fogelman45 demonstrated the existence of similarly heterogeneous LDL fusion products in early lesions during development of experimental, diet-induced atherosclerosis. If the LDL derivatives observed by Frank and Fogelman are identical to E-LDL, as we propose, then all subfractions are present in vivo. Thus, assessing the biological properties of each subfraction, although of interest, is not of prime importance to the formulation of our working hypothesis. We propose that E-LDL activates complement and thus generates chemotactic peptides, causing a first influx of blood monocytes and perhaps granulocytes into the lesion. Granulocytes are short-lived, but the monocytes differentiate to macrophages, upregulate their scavenger receptors, and ingest the E-LDL. This event is accompanied by massive release of MCP-1, which attracts additional monocytes to the lesion. If the transport system becomes overloaded, the macrophages become immobile, liberation of proinflammatory cytokines may attain significance, and some foam cells may die, releasing their intracellular content of lipid, which causes renewed complement activation. A vicious circle of events is thus promoted, providing a quite simple explanation for the chronic inflammation that is characteristic of the atherosclerotic lesion.
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (SFB project 311, D10 grant to S.B.). We thank Petra Jenichen for sequencing and the nurses of the university blood bank, in particular Anna Bacic and Marina Glück, for providing us with fasting plasma and buffy coats.
Received November 27, 1997; accepted March 16, 1998.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Ylä-Herttuala S, Lipton BA, Rosenfeld ME,
Sarkioja, T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D.
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