23
Inhibition of Copper-Mediated Oxidation of
LDL by Rat Serosal Mast Cells
A Novel Cellular Protective Mechanism Involving Proteolysis
of the Substrate Under Oxidative Stress
Ken A. Lindstedt, Jorma O. Kokkonen, and Petri T. Kovanen
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Rat serosal mast cells, when stimulated to exocytose their cytoplasmic granules, effectively blocked the
copper-mediated oxidation of low density lipoproteins (LDLs) in vitro. This effect depended on the
proteolytic activity of the formed extracellular granule remnants, since specific inhibition of chymase, the
neutral protease that they contain, blocked the protective effect of the mast cells. The mechanism of this
chymase-mediated inhibition of LDL oxidation was found to be binding of the copper ions present in the
incubation medium by peptides released from LDL on proteolytic degradation of their apolipoprotein B
(apoB) component. This was verified by demonstrating that addition of such peptides to LDL-copper ion
mixtures completely prevented oxidation of LDL and that this protective effect could be overcome by
adding copper ions in excess. Furthermore, proteolytic degradation of the apoB of LDL, with concomitant
release of copper-containing peptides, left the partially degraded apoB without the copper ions necessary
for propagation of LDL oxidation. These observations provide the first evidence for cell-mediated
inhibition of LDL oxidation. (Arteriosclerosis and Thrombosis 1993;13:23-32)
KEYWORDS • chymase • exocytosis • granules • atherosclerosis
T
he cholesterol found in atherosclerotic lesions is
derived mainly from low density lipoproteins
(LDLs) that have penetrated into the intima,
the inner layer of the arterial wall.1 Native LDLs are not
effectively metabolized by macrophages, which are the
precursors of most of the cholesteryl ester-filled foam
cells found in atherosclerotic lesions.2 Therefore, it has
been postulated that LDL must be first modified in the
intima to be recognized and taken up by macrophages.3-5
One cell type present in atherosclerotic lesions in
both animals and humans is the mast cell.67 Recent
studies with rat serosal mast cells both in vitro and in
vivo have disclosed a specific mechanism by which mast
cells may modify LDL and render it susceptible to
uptake by macrophages.8-12 The mast cell-induced uptake of LDL by the macrophages is initiated by a strictly
ordered sequence of events in which stimulation of mast
cells first leads to exocytosis of their cytoplasmic granules, followed by binding of LDL to the heparin proteoglycan component of the formed extracellular granule
remnants and proteolytic modification of the granule
remnant-bound LDL by the granule-neutral proteases,
chymase and carboxypeptidase A. The two granule
remnant-bound neutral proteases act in sequence: chymase first cleaves peptides from the apolipoprotein
(apo) B, and then carboxypeptidase A cleaves the
carboxy-terminal aromatic or branched-chain aliphatic
amino acids from the peptides so formed.13 The peptides cleaved by chymase and liberated from LDL
From the Wihuri Research Institute, Helsinki.
Address for correspondence: Dr. Petri T. Kovanen, Wihuri
Research Institute, Kalliolinnantie 4, SF-00140 Helsinki, Finland.
Received June 11, 1992; revision accepted August 24, 1992.
particles are a heterogeneous group of relatively small
peptides having Mrs of less than 5,000. The result of the
binding and degradation of LDL by mast-cell granule
remnants is a modified LDL particle characterized by
selective loss of protein and increased particle size from
20 nm up to 100 nm through fusion.9 Finally, granule
remnants loaded with the modified LDL particles are
phagocytosed by macrophages, within which the cholesteryl ester droplets characteristic of foam cells are
formed.
Another type of lipoprotein modification, oxidation,
appears to play a major role in generating a modified
LDL that leads to foam cell formation in atherosclerotic
lesions in both animal models and humans.14-16 Experiments conducted in vitro have demonstrated that LDL
may be oxidized by many types of intimal cells, e.g.,
endothelial cells,17 smooth muscle cells,18 or macrophages.19 The oxidized LDL present in atherosclerotic
lesions may be formed by several mechanisms: release
of superoxide anions from the cells,20 direct action of
membrane-bound enzymes on LDL,21 or transfer to
LDL of lipid peroxides generated within cell membranes by such enzymes as cellular lipoxygenases.22
After the initial "seeding" of LDL with peroxides,
ions of transition metals such as copper and iron have
been shown to play a crucial role in the further oxidative
modification of LDL.23 Indeed, the prototype of oxidized LDL has been "copper-oxidized LDL," a modification generated in vitro in the absence of cells. More
recently, it has been realized that only if ions of either
copper or iron are present at nanomolar concentrations
will the cell-mediated oxidation of LDL be of sufficient
magnitude to induce uptake of the modified LDL
24
Arteriosclerosis and Thrombosis Vol 13, No 1 January 1993
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particles by macrophages.24 Most importantly, it has
been reported that the LDL particles themselves are
able to bind three to eight copper ions,25 suggesting the
presence in vivo of LDL-copper ion complexes.
Mast cells may also be involved in the oxidative
metabolism of LDL. The mast cells, on stimulation,
secrete superoxide anions.26 However, at the same time
they secrete a superoxide-scavenging system composed
of secretory granule-bound superoxide dismutase
(SOD) and peroxidase.27 These earlier observations
suggested that mast cells were potential candidates for
involvement in the oxidative metabolism of LDL as
either prooxidants or antioxidants. To study the role of
these cells in the oxidation of LDL, we incubated LDL
with copper ions in the presence of rat serosal mast
cells. In contrast with the other intimal cell types
studied so far, mast cells totally inhibited copper ionmediated oxidation of LDL. Unexpectedly, these experiments disclosed that the mechanism for inhibition of
the copper-mediated LDL oxidation was the proteolytic
degradation of the apoB moiety of LDL by granule
remnants exocytosed from stimulated mast cells.
Methods
Materials and Animals
Sodium [125I]iodide (13-17 mCi/jug) and [U-14C]sucrose
(>350 Ci/mmol) were purchased from Amersham International. Compound 48/80, diethyldithiocarbamic acid
(DDC), chymotrypsin, and bovine serum albumin were
obtained from Sigma. Human serum albumin was purchased from Kabi Diagnostica. Phenylmethylsulfonyl fluoride (PMSF) was obtained from Boehringer Mannheim.
Malonaldehyde bis(dimethyl acetal) was purchased from
Aldrich-Chemie. 2-Thiobarbituric acid was obtained from
Fluka. Copper (II) sulfate pentahydrate was purchased
from Merck. Dulbecco's phosphate-buffered saline (PBS)
was obtained from GIBCO. Male Wistar rats (200-500 g)
and female NMRI mice (25-35 g) were purchased from
Orion (Espoo, Finland).
Preparation and Labeling of Lipoproteins
Human LDLs (d= 1.019-1.050 g/ml) were isolated
from plasma by sequential ultracentrifugation,9 iodinated by the iodine monochloride method as previously
described,28 and labeled with [U-14C]sucrose as described by Pittman et al.29 To obtain EDTA-free LDL,
either labeled or unlabeled LDLs were dialyzed against
three changes (5 1 each) of buffer A (150 mM NaCl and
5 mM tris(hydroxymethyl)aminomethane [Tris]-chloride, pH 7.4). In experiments the labeled lipoproteins
were diluted with unlabeled LDL to give the specific
activities indicated in the figure legends. The amounts
and concentrations of LDL are expressed in terms of
protein.
Isolation and Treatment of Mast Cell Granules
Mast cells and mast cell granules were isolated as
described.30 Briefly, cells were collected from the peritoneal and pleural cavities of rats by lavage with isolation buffer (PBS supplemented with 0.5 mg/ml bovine
serum albumin and 5.6 mM glucose, pH 7.3). The cells
were washed and resuspended in RPMI 1640 supplemented with 10 mg/ml human serum albumin, 5% fresh
rat serum, 25 mM NaCl, 100 IU/ml penicillin, and 2 mM
L-glutamine. The cells were seeded into plastic Petri
dishes and incubated in a humidified CO2 (5% CO2 in
air) incubator at 37°C for 1 hour. Nonadherent cells,
chiefly mast cells (90-95% purity), were removed for
use in experiments. Mast cell granules were then isolated by stimulating the cells at 37°C for 15 minutes with
compound 48/80, a positively charged small-molecularweight compound that causes specific and noncytotoxic
degranulation of mast cells. The supernatant, containing the secreted granules, was removed and centrifuged
at 12,000g for 15 minutes to sediment the granule
remnants. Proteolytically inactive granule remnants,
i.e., PMSF-treated granule remnants, were prepared as
described.8 The quantity of granule remnants is expressed in terms of granule remnant protein.
Purification of Mast-Cell Granule Chymase and
Heparin Proteoglycan
Chymase from rat serosal mast cells was purified by
hydrophobic chromatography on octyl-Sepharose
CL-4B as described by Kido et al.31 Mast-cell granule
heparin proteoglycan was purified by treating the soluble proteoglycan fraction released on mast cell stimulation with chondroitinase ABC as described:32 After
incubation at 37°C for 18 hours the heparin proteoglycan was dialyzed against three changes (5 1 each) of
buffer A. The amount of heparin proteoglycan was
determined by the Alcian blue method, with commercial
heparin as the standard.33
Isolation of Mouse Peritoneal Macrophages
Macrophages were harvested from unstimulated NMRI
mice in PBS containing 1 mg/ml bovine serum albumin as
described by Goldstein et al.3 The peritoneal cells were
resuspended in RPMI 1640 culture medium containing
10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 jug/ml streptomycin. The cells were seeded
into plastic Petri dishes (35x10 mm, Falcon) and incubated in a humidified CO2 incubator (5% CO2 in air) at
37°C for 1 hour. Three rinses of PBS (2 ml) were used to
remove nonadherent cells. For experiments the dishes
received 1 ml culture medium (RPMI 1640 with L-glutamine containing 10% fetal calf serum, 100 IU/ml penicillin, and 100 /xg/ml streptomycin).
Preparation of LDL Degradation Products
LDL (500 ixg) was incubated in 300 /AI buffer A
containing 20 /xg granule remnants at 37°C for 18 hours.
An aliquot was treated with trichloroacetic acid to
determine the degree of proteolytic degradation of
LDL.28 The low-molecular-weight degradation products
were separated from the proteolyzed LDL particles and
granule remnants by ultrafiltration through a Centricon
10 filter (Amicon) with a 10,000-d cutoff point. For this
purpose the filter was centrifuged four times at l,400g
for 30 minutes, and the ultrafiltrates were pooled and
analyzed for protein concentration.
Copper-Mediated Oxidation of LDL
In a standard assay EDTA-free LDL (100 jtig) was
incubated in 300 /xl PBS containing 7 fjM CuSO4 or
mast cell-derived material at 37°C for 3 hours. The
extent of lipid peroxidation of LDL was measured as
thiobarbituric acid (TBA)-reactive materials and expressed as malondialdehyde (MDA) equivalents.34
Lindstedt et al
Inhibition of LDL Oxidation by Mast Cells
25
Mast Cell-Mediated Inhibition of LDL Oxidation
In a standard assay, mast cells (106) in 250 /xl PBS
were stimulated with compound 48/80 (5 tig/ml) for 5
minutes at 37°C. After incubation the cells were centrifuged at 150g for 10 minutes. The supernatant containing the exocytosed material was removed (150g supernatant). The cell pellet was washed twice and
resuspended in 250 id PBS. Aliquots of the exocytosed
material (the 150g supernatant) derived from 4xlO 3 1 x 105 mast cells and the corresponding cell pellets were
then tested for their effects on the copper-mediated
oxidation of LDL as described above.
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Uptake of Modified LDL by Mouse
Peritoneal Macrophages
In a standard assay, [U14C]sucrose-LDL (100 iig) was
incubated in 300 /i,l PBS containing 7 tiM CuSO4 and 10
iu,g untreated or PMSF-treated granule remnants at
37°C for 3 hours. After incubation the granule remnants
were sedimented by centrifugation at 12,000g for 15
minutes. The supernatant was removed, and a 100-fil
sample was analyzed for the presence of TBA-reactive
material and a 10 -/xl sample for electrophoretic mobility. The remaining material was dialyzed against two
changes (5 1 each) of buffer (150 raM NaCl, 5 mM
Tris-chloride, and 1 mM EDTA, pH 7.4) and applied to
the macrophage dishes. After incubation for 18 hours
the uptake of [14C]sucrose-LDL was determined as
described.35 Uptake is expressed as micrograms of LDL
protein per milligram of cell protein.
Determination of Copper Ions in
the Reaction Mixture
The concentration of copper ions was determined by
atomic absorption spectrophotometry (Perkin Elmer
A AS-5000) by using the graphite furnace method according to the manufacturer's recommendations or according to the DDC method described by Marciani et
al.36 For analysis with the latter method, samples or
copper sulfate standards (0-100 fiM) were mixed into
400 /xl buffer A containing 1.5 /xM DDC. The absorbance of the copper-DDC complex was measured with
an automatic microplate reader (Multiskan MCC, Labsystems) at 450 nm. In an additional experiment we
used atomic absorption spectrophotometry to measure
the concentration of copper ions in the granule-remnant
fraction and found that the granule remnants were
unable to bind copper ions (data not shown).
Other Assays
Protein was determined by the procedure of Lowry et
al37 with bovine serum albumin as the standard. The
electrophoretic mobility of the oxidized LDL was determined on cellulose acetate plates (Helena Laboratories) stained with Ponceau red.
Results
The effect of mast cells on the copper-mediated
oxidation of LDL was measured by incubating LDL with
copper ions in the presence of either unstimulated mast
cells or mast cells that were stimulated with compound
48/80. The formation of TBA-reactive material in the
incubation medium was taken as an index of lipid
peroxidation of LDL. In the presence of unstimulated
1
-48/80
- Cu 2+
2
-48/80
+ Cu 2+
3
4
+48/80
+48/80
+ Cu 2+ - Cu 2+
FIGURE 1. Bar graph shows the effect of stimulated rat
serosal mast cells on copper-mediated oxidation of low density
lipoprotein (LDL). Mast cells (105) were incubated at 37°C in
300 id Dulbecco's phosphate-buffered saline containing 100
/xg EDTA-free LDL (column 1) and the following additions:
7 fiM CuSO4 (column 2); 7 \iM CuSO4 and 5 /jg/ml
compound 48180 (column 3); or 5 fig/ml compound 48/80
(column 4). After incubation for 3 hours the amount of
thiobarbituric acid (TBA) -reactive material was determined
as described in the text. Each value is the average of duplicate
incubations. MDA, malondialdehyde.
mast cells, copper ions caused rapid and extensive
oxidation of LDL (Figure 1, column 2). In contrast, the
copper-induced oxidation of LDL was totally abolished
if the mast cells were stimulated at the beginning of
incubation (column 3). A control experiment showed
that in the absence of copper ions, stimulated mast cells
themselves did not trigger the formation of TBA-reactive material (column 4).
To study whether the capacity of stimulated mast
cells to protect against oxidation of LDL resided in the
cells or in the material released from them on stimulation, we sedimented the stimulated mast cells by lowspeed centrifugation and made separate studies of the
effects of the cell pellet and the supernatant on the
copper-mediated oxidation of LDL. As shown in Figure
2, the protective effect resided in the supernatant, which
contained the material exocytosed from the stimulated
mast cells. In the incubation system containing 330
/xg/ml LDL protein and 7 ju,M CuSO4, the exocytosed
material caused a concentration-dependent inhibition
of LDL oxidation, 85% inhibition being achieved with
25 /xl exocytosed material, corresponding to the amount
of material released from approximately 100,000 mast
cells.
To locate the protective agent present in the material
released from stimulated mast cells (150g supernatant),
the granule remnants were sedimented by centrifugation of the supernatant at 12,000g.32 The two fractions
formed (granule remnants and granule-free supernatant) were then incubated separately with LDL in the
presence of copper sulfate. It was found that most of the
protective capacity was located in the granule-remnant
fraction (Figure 3). Indeed, addition of increasing vol-
26
Arteriosclerosis and Thrombosis
Vol 13, No 1 January 1993
c- 5 °
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Q)
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15
20
25
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. A 150 xg supernatant
O granule-free supernatant
A granule remnants
10 H
Medium added (nl / tube)
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FIGURE 2. Line graph shows inhibition of copper-mediated
oxidation of low density lipoprotein (LDL) by material exocytosedfrom mast cells. Mast cells (106) were preincubated in
300 id Dulbecco's phosphate-buffered saline (PBS) at 37°C
for 15 minutes. The cells were stimulated with compound
48/80 (5 fig/ml) for 5 minutes. Cells were then sedimented by
centrifugation at 150% for 10 minutes. The exocytosed material (150g supernatant) was removed, and the cell pellet was
washed twice and resuspended in 300 id PBS. The indicated
amounts of mast cell pellet (A) and 150g supernatant (•) were
then incubated in a final volume of 300 id PBS containing 100
fig LDL and 7 iiM CuSO4. After incubation at 37°C for 3
hours, the amount of thiobarbituric acid (TBA) -reactive material was determined as described in the text. Each value is the
average of duplicate incubations. MDA, malondialdehyde.
umes of either granule remnants or the 150g supernatant, from which the granule remnants were isolated,
yielded identical results.
The two major components of the granule remnants
of rat serosal mast cells are heparin proteoglycans and
the neutral protease chymase38; the former binds apoB
and the latter degrades it.10 We isolated and purified
both components of the granule remnants for separate
studies of their effects on the copper-induced oxidation
of LDL. We found that heparin proteoglycans did not
prevent the formation of TBA-reactive material (Figure
4). In contrast, addition of increasing amounts of purified chymase inhibited the oxidation of LDL in a
concentration-dependent fashion, maximal inhibition
being achieved with approximately 6 /xg/ml purified
chymase.
To test whether the protective capacity of the granule
chymase was due to its proteolytic action on the apoB
component of LDL, chymase was specifically inhibited
by treating the granule remnants with PMSF.8 As shown
in Figure 5A, incubation of LDL with copper ions
caused extensive oxidation of LDL, i.e., more than a
10-fold increase in the concentration of TBA-reactive
material (columns 1 versus 2). The copper-induced LDL
oxidation could be inhibited by adding granule remnants to the incubation system (column 3). In contrast,
if the granule remnants were first treated with PMSF
and then added to LDL along with copper sulfate, no
such inhibition of LDL oxidation could be observed
(column 4). After the incubations, aliquots were taken
from the incubation mixtures to determine the electrophoretic mobilities of the LDL particles. As shown in
0
\
•
i
i
0.5
1.0
1.5
I
2.0
*
i
2.5
Medium added (ul)
FIGURE 3. Line graph shows inhibition of copper-mediated
oxidation of low density lipoprotein (LDL) by mast cell
granules. Mast cells (2xlO6) were preincubated in 100 id
Dulbecco's phosphate-buffered saline (PBS) for 15 minutes
and then stimulated with compound 48/80 (5 ng/ml) for 5
minutes. The cells were sedimented by centrifugation at 150g
for 10 minutes, and the 150g supernatant was removed. One
half of the 150g supernatant (50 id) was further centrifuged
at 12,000gfor 15 minutes to sediment the granule remnants.
The 12,000g supernatant, i.e., the granule-free supernatant,
was removed, and the granule remnants were resuspended in
50 /d PBS. Indicated amounts of each fraction, i.e., 150g
supernatant ( A ) , granule remnants ( A ) , and granule-free
supernatant (o), were incubated in a final volume of 300 id
PBS containing 100 fig LDL and 7 fiM CuSO4. After
incubation at 37°C for 3 hours, the amount of thiobarbituric
acid (TBA)-reactive material was determined as described in
the text. Each value is the average of duplicate incubations.
MDA, malondialdehyde.
the inset of Figure 5A, the LDL incubated with PMSFtreated granule remnants and copper ions showed the
increased electrophoretic mobility typical of oxidized
LDL (lanes 4 and 2, respectively). On the other hand,
the LDL incubated with proteolytically active granule
remnants and copper ions showed electrophoretic mobility similar to that of untreated native LDL (lanes 3
and 1, respectively). The effect of the granule remnants
on oxidative modification of LDL was also studied with
a bioassay in which the ability of cultured mouse
peritoneal macrophages to internalize modified LDL
was tested.3 As shown in Figure 5B, oxidized LDL was
taken up avidly by mouse peritoneal macrophages (column 2). Addition of proteolytically active granule remnants to the incubation system totally blocked the
copper-induced increment in LDL uptake, so that such
LDLs were taken up by macrophages at approximately
the same very low rate as native LDL (columns 3 and 1,
respectively). Finally, inhibition of chymase activity with
PMSF blocked the protective effect of the granule
remnants and thus permitted the copper-mediated oxidation of LDL, leading to increased uptake of LDL by
macrophages (column 4).
Lindstedt et al
100
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ffl
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75
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Inhibition of LDL Oxidation by Mast Cells
27
O
Heparin proteoglycan
50
25
Chymase
12
Inhibitors (|ig / ml)
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FIGURE 4. Line graph shows inhibition of copper-mediated
oxidation of low density lipoprotein (LDL) by mast cellgranule chymase. Mast cell-granule chymase and heparin
proteoglycan were purified as described in the text. Indicated
amounts of heparin proteoglycan (o) and chymase (•) were
incubated in 300 /J Dulbecco's phosphate-buffered saline
containing 100 /ig LDL and 14 /xM CuSO4 at 37°C for 3
hours. The amount of thiobarbituric acid (TBA)-reactive
material was determined as described in the text. The 100%
control values for heparin proteoglycan and chymase were
19.5 and 24.0 nmol malondialdehydelmg LDL protein, respectively. Each value is the average of duplicate incubations.
To quantitatively relate the protective effect of chymase to its ability to proteolyze the apoB component of
LDL, we measured the formation of TBA-reactive
material as a function of the degree of apoB degradation. For this purpose, LDLs were first incubated in the
presence of granule remnants for various periods of
time, after which the proteolytic degradation of apoB
was stopped by addition of PMSF. Oxidation of the
granule remnant-treated LDL was then initiated by
addition of copper sulfate, and the amounts of TBAreactive material were measured after a standard incubation of 3 hours. As shown in Figure 6A, incubation of
LDL in the presence of granule remnants resulted in
progressive degradation of apoB, measured as production of trichloroacetic acid-soluble material. It was
found that the more extensively apoB was proteolyzed,
the less susceptible it was to oxidation (Figure 6B).
Indeed, after degradation for 60 minutes, LDL was
practically resistant to copper-mediated oxidation.
The above result suggests that either the degradation
products, i.e., the peptides and amino acids released
from apoB,13 possessed a protective effect or that the
proteolytically degraded LDL particles themselves are
more resistant to oxidation than are native LDLs. The
first alternative was tested by measuring the protective
effect of the proteolytic degradation products released
from apoB. For this purpose LDL was incubated with
granule remnants, and the low-molecular-weight degradation products formed were separated from the proteolyzed LDL particles by ultrafiltration through a filter
with a 10,000-d cutoff point. Increasing amounts of the
degradation products were then added to a reaction
4
+ Cu2+
+ gran
+ PMSF
FIGURE 5. Bar graphs show effect of chymase inhibition on
mast cell granule-mediated inhibition of low density lipoprotein (LDL) oxidation. f'"C]Sucrose-LDL (100 ng; 3,970
disintegrations per minute//jgprotein) was incubated in 300 \d
Dulbecco's phosphate-buffered saline either alone (column 1
and lane 1 in the inset), with 7 ixM CuSO4 (column 2 and lane
2 in the inset), with 7 fiM CuSO4 and 10 ixg native granule
remnants (column 3 and lane 3 in the inset), or with 7 fiM
CuSO4 and 10 (ig phenylmethylsulfonyl fluoride (PMSF)treated granule remnants (column 4 and lane 4 in the inset) at
37°Cfor 3 hours. Incubations were stopped by sedimenting the
granule remnants by centrifugation at 12,000gfor 15 minutes.
The granule-free supernatant was then removed and analyzed
for thiobarbituric acid (TBA)-reactive material (panel A) or
electrophoretic mobility (inset) as described in the text. In the
experiment with macrophages (panel B) each monolayer
received 1 ml culture medium containing 60 fjg [I4C]sucroseLDL. After incubation for 18 hours the uptake of f'4CJsucrose-LDL was determined as described in the text. Each
value is the average of duplicate incubations. MDA, malondialdehyde; n-LDL, native LDL; gran, granule remnants.
mixture containing incubation medium, intact LDL
particles, and copper sulfate. It was found that the
degradation products of apoB prevented the coppermediated oxidation of LDL in a dose-dependent fashion
(Figure 7A). Addition of copper sulfate in excess overcame the protective effect of the proteolytic degradation
products (Figure 7B). It could be calculated that 15
28
Arteriosclerosis and Thrombosis
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40
Vol 13, No 1 January 1993
60
Incubation time (mln)
FIGURE 6. Line graphs show inhibition of copper-mediated
low density lipoprotein (LDL) oxidation as a function of LDL
degradation. I25I-LDL (100 ng; 1,800 counts per minute/tig
protein) was incubated at 37°C in 100 yl Dulbecco's phosphate-buffered saline (PBS) containing 10 fig granule remnants for the indicated time intervals. Proteolysis was stopped
by adding phenylmethylsulfonyl fluoride (final concentration,
250 fjg/tnl). A sample of each reaction mixture was taken to
determine the amount of I25I-labeled trichloroacetic acidsoluble degradation products (panel A) as described in the
text. Another sample of each reaction mixture was incubated
in a final volume of 300 \J1 PBS containing 7 pM CuSO4.
After incubation at 37°C for 3 hours, the amount of thiobarbituric acid (TBA) -reactive material was determined (panel
B) as described in the text. Each value is the average of
duplicate incubations. MDA, malondialdehyde.
fig/ml apoB degradation products was required for
inhibition of the oxidation of 330 /ig/ml LDL induced by
13 JAM CUSO 4 in the given buffer.
The stoichiometry between the apoB peptides and
copper sulfate in the above experiment suggested direct
interaction between the divalent copper ions and the
peptides. This hypothesis was tested by investigating the
effect of apoB peptides on the elution of copper sulfate
on a Bio-Gel P-2 column. As shown in Figure 8A, a
control solution containing copper sulfate alone eluted
at the position corresponding to the total volume of the
column. In contrast, if the peptides produced by proteolytic degradation of apoB by chymase were incubated
with copper sulfate and the reaction mixture was chro-
matographed on a Bio-Gel P-2 column, part of the
copper ions coeluted with these peptides (Figure 8B-C,
fractions 10-38), indicating that the apoB-derived peptides bound copper ions.
The second possibility for the protective effect of
apoB degradation is that the release of copper-containing peptides from apoB on proteolytic degradation of
LDL lowers the copper ion content of the apoB, thereby
rendering LDL resistant to oxidation. This hypothesis
was tested as follows: LDL (400 /xg) was incubated in
400 fi\ buffer A containing 20 /xM CuSO4 at 37°C for 15
minutes. Unbound copper ions were separated from
LDL-bound copper ions by ultrafiltration. The remaining LDLs were resuspended in buffer A; analysis
showed they contained approximately two thirds of the
added copper ions. These copper-containing LDLs (100
/ig) were incubated in 300 fi\ buffer A with or without 5
fig purified chymase at 37°C for 18 hours. The peptides
formed were separated from the degraded LDL by
ultrafiltration. Protein and copper ion concentrations in
the degraded copper-containing LDL fraction and in
the ultrafiltrate containing the degradation products
were determined. Treatment with chymase led to the
release of not only low-molecular-weight degradation
products of apoB (43% of total apoB mass) but also to
substantial (87%) amounts of the LDL-bound copper
ions into the incubation medium, thus producing copper-deficient LDL particles (Table 1).
Finally, we tested whether the proteolytic modification of LDL by chymase treatment would render the
LDL particles resistant to oxidation. For this purpose,
copper-containing LDLs were prepared by first incubating LDLs with copper sulfate and then removing unbound copper ions as described above. Subsequently,
the copper-containing LDL particles were incubated at
37°C with purified mast cell chymase up to 6 hours. As
shown in Figure 9A, such incubation led to progressive
degradation of apoB. More importantly, the proteolyzed LDL, i.e., the copper-deficient LDL particles, did
not become oxidized like the LDLs that were not
incubated with chymase and, consequently, contained
all the copper ions bound to them during the initial
incubation with copper sulfate (Figure 9B). However,
addition of copper sulfate (7 j^M) to the proteolyzed
copper-deficient LDL particles resulted in formation of
TBA-reactive material in amounts that equaled those
observed when copper sulfate was added to untreated
LDL (data not shown).
Discussion
The present in vitro study demonstrates that stimulation of rat serosal mast cells leads to inhibition of
copper-mediated oxidation of LDL. This inhibition depends on the extracellular generation of copper-binding
apoB peptides from LDL by chymase, the neutral
protease of mast cells. Since chymase is a matrix-bound
component of the cytoplasmic granules of mast cells,
this enzyme does not gain access to the apoB component of LDL present in the extracellular fluid unless the
granules are exocytosed in response to mast cell stimulation. In previous studies, we have shown that even
after exocytosis the granule-bound chymase does not
degrade all of the LDL particles present in the incubation medium, but only the very small subfraction of the
particles that are bound by the granule remnants.11
Lindstedt et al
Inhibition of LDL Oxidation by Mast Cells
29
ID
6
9
12
Degradation products
15
10
15
20
25
Excess copper sulfate
(M9' ml)
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FIGURE 7. Line graphs show inhibition of low density lipoprotein (LDL) oxidation by LDL degradation products. LDL
degradation products were obtained as described in the text. Each reaction mixture (panel A) contained 300 fd Dulbecco's
phosphate-buffered saline, 100 i*g LDL, 13 fiM CuSO4, and the indicated amounts of degradation products. After incubation at
37°Cfor 3 hours, the amount of thiobarbituric acid (TBA)-reactive material was determined as described in the text. In a control
experiment (panel B), 100 fjg LDL was incubated in 300 fd PBS containing 13 \iM CuSO4, 5 fxg degradation products, and the
indicated concentrations of excess copper sulfate. After incubation at 37°Cfor 3 hours, the amount of TBA-reactive material was
determined as described in the text. The 100% control values in Figures 7A and 7B were 26.2 and 33.9 nmol malondialdehydelmg
LDL protein, respectively. Each value is the average of duplicate incubations.
These remnant-bound particles appear to form a barrier
that shields the enzyme molecules from contact with the
apoB of the unbound LDL particles.12 Accordingly, only
a small fraction of the apoB molecules present in the
incubation system are degraded by the granule remnants and act as the source of the copper-binding
peptides. Nevertheless, the quantity of peptides released was found to be sufficient to protect both the
bound and unbound fractions of LDL from coppermediated oxidation.
The peptides released from the apoB portion of
granule-remnant-bound LDL on proteolytic degradation by chymase have been found to have molecular
weights of less than 5,000 d.13 The present report on the
ability of these small peptides to bind copper ions is the
first biological function assigned to them. The mechanism by which these peptides inhibit the copper-induced
oxidation of LDL was found to be the binding of copper
ions present in the aqueous phase, with formation of
apparently redox-inactive copper ion-peptide complexes. The amino acids histidine, cysteine, and tryptophan have been shown to form complexes with copper
ions.39-41 Since these amino acids are among the peptides released from the apoB of LDL by the proteolytic
action of mast-cell granule remnants,13 it appears likely
that they were the agents responsible for the observed
binding of copper ions. However, isolation and sequencing of the apoB peptides released by chymase are
required to identify the copper-binding sites on apoB.
Furthermore, amino acids such as histidine and tryptophan are also strong inhibitors of copper-mediated
LDL oxidation, suggesting that other proteolytic peptides besides those derived from the apoB of LDL could
act as copper-ion chelators. Indeed, we have shown that
high density lipoprotein degradation products are capa-
ble of inhibiting the copper-mediated oxidation of LDL
(P.T. Kovanen et al, unpublished observations).
Copper-induced oxidation of LDL was also prevented
by another mechanism. During chymase-mediated degradation of LDL, the gradual proteolytic cleavage of
apoB led to the release not only of peptides but also of
copper ions. Since the released peptides had an affinity
for copper ions, it is conceivable that the copper ions,
known to have affinity for apoB,42 had initially been
bound to the peptide segments and were then released
passively along with them. Thus, the second protective
mechanism was the generation of proteolytically modified, copper-deficient LDL particles. However, since
chymase is a granule remnant-bound protease and only
the granule remnant-bound LDL particles are degraded by chymase, only a minor subtraction of LDL
could potentially benefit from this type of protection
against copper-catalyzed oxidation.
The major protective mechanism of mast cells against
copper-mediated oxidation of LDL could be assigned to
the proteolytic action of granule-remnant chymase.
However, a slight inhibition of LDL oxidation was still
observable after the granule remnants (and chymase)
had been removed from the mast cell-conditioned
medium (Figure 3, granule-free supernatant). This
granule-free fraction of the medium contains all the
histamine released from the exocytosed mast cell granules.38 When copper ions are immobilized in chelating
Sepharose beads, a column of such Sepharose is then
able to adsorb histamine from the eluent.43 This ability
of histamine and copper ions to form complexes is not
surprising, since histamine contains the ring structure of
histidine that is responsible for the binding of copper
ions.39 Thus, copper ions are effectively bound not only
by the apoB-derived peptides but also by histamine,
which likewise protects LDL from oxidation. Indeed, by
30
Arteriosclerosis and Thrombosis
Vol 13, No 1 January 1993
TABLE 1. Binding of Copper Ions to LDL and Release of
Copper-Containing Peptides by Granule Chymase
0.40
Fraction
a> §
o s
0.30
LDL degradation products
Proteolyzed LDL
LDL
o E
0.20
C u
to S
0.10
0.15
1
0.05
2500
;o
^^
2000
(cpm i/irac
m
a>
1500
a. c
o> o
a. s
Mabelled
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
0.10
in
CM
Copper
(/imol/1)
2.80 (87)
0.42 (13)
3.22 (100)
LDL, low density lipoprotein. The values for LDL are calculated values and represent the sums of LDL degradation products
plus proteolyzed LDL. Each value is the average of duplicate
incubations. Values in parentheses represent percentages of control values. For further details, see "Methods."
<s
2 S
Protein
(mg/1)
212 (43)
286 (57)
498 (100)
1000
500
10
20
30
40
50
60
Fraction number
FIGURE 8. Line graphs show binding of copper ions by
apolipoprotein B-derived peptides. Low density lipoprotein
degradation products were obtained as described in the text.
Copper sulfate (1 mM) was incubated either alone (panel A)
or with 100 yg 125I-labeled peptides (383 counts per minute/
fig) (panels B and C) in 200 fJ buffer A at 37°C for 15
minutes. After incubation, the reaction mixtures were applied
to a Bio-Gel P-2 column (1.0x17 cm). The column was
eluted with buffer A at a flow rate of 4 ml/hr at room
temperature. Fractions of 400 fxl were collected into tubes
containing 25 \il of 25 mM diethyldithiocarbamic acid. The
fractions were measured for their copper ion concentration
and I25I radioactivity as described in the text. The void volume
(Vo) and the total volume (V,) of the column are indicated by
arrows (panel A). Note the different scales in absorbance in
panels A and B.
adding sufficient commercial histamine to mixtures of
copper ions and LDL, we could inhibit oxidation of
LDL completely (data not shown).
The cytoplasmic secretory granules of mast cells
contain SOD, another matrix-bound enzyme and a
potent scavenger of superoxide anions.26'27 Therefore,
this granule enzyme must also be regarded as a potential candidate for the protective defense system of
stimulated mast cells. PMSF, a specific inhibitor of the
granule chymase that had no effect on the SOD activity
of granule remnants (data not shown), inhibited the
protective effect of mast-cell granule remnants by about
80% (Figure 5). Whether the residual inhibition of LDL
oxidation (approximately 20%; 0-20% in different experiments) observed with granule remnants was due to
an SOD effect remains to be shown. It is noteworthy
that Parthasarathy and coworkers22 have shown that
human recombinant SOD effectively inhibits copperinduced oxidation of LDL. In addition, these workers
have found that SOD effectively blocks oxidation of
LDL by smooth muscle cells.22 Stimulated mast cells
could be able, by means of their granule SOD, to
effectively inhibit cell-mediated oxidation of LDL, notably oxidation by smooth muscle cells and monocytes,
in which the superoxide pathway predominates.2044
In terms of human disease, oxidation of LDL in the
arterial intitna, the site of atherogenesis, appears to be
of major importance.4 Thus, the various cell types of the
arterial intima studied so far, i.e., the endothelial cell,
monocyte-macrophage, and smooth muscle cell, all have
the ability to oxidize LDL in vitro. The mast cell, a cell
type also present in the arterial intima, clearly seems to
be an exception among the intimal cells. Indeed, the
mast cell is the first cell type of the arterial intima
studied that prevents rather than promotes oxidation of
LDL. This unique property of mast cells is a consequence of their ability to induce extracellular degradation of proteins that have an affinity for heparin proteoglycan such as apoB, an ability that appears not to be
shared by the other cell types present in the arterial
intima.
Initiation of the oxidative modification of LDL in the
arterial intima may result both from cellular oxidative
reactions and from cell-independent spontaneous oxidative reactions in the extracellular fluid.4-24 After initiation, the propagation of the lipid peroxidation necessary to modify LDL to a form that can be recognized by
the scavenger receptors of macrophages is greatly facilitated by trace amounts of transition metal ions. In the
presence of such metal ions, stimulation of mast cells
with ensuing apoB degradation and release of histamine
Lindstedt et al Inhibition of LDL Oxidation by Mast Cells
31
References
•=•
20
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Incubation time (hours)
FIGURE 9.
Line graphs show inhibition of oxidation of
copper-containing low density lipoprotein (LDL) by granule
chymase. I2SI-LDL (1.5 mg; 2,100 counts per minute1fig) was
incubated in 3 ml buffer A containing 20 JXM CuSO4 at 37°C
for 15 minutes. Unbound copper ions were separated from
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text. Copper-containing LDL (100 fig) was then incubated in
300 fd buffer A with or without 2.5 fig granule chymase at
37°C for the indicated times, after which the amounts of
trichloroacetic acid-soluble (panel A) and thiobarbituric
acid (TBA) -reactive (panel B) materials were determined as
described in the text. Each value is the average of duplicate
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LDL in the arterial intima? Current evidence for the
presence of oxidized LDL in atheromatous lesions of
the arterial intima45 does not favor this possibility.
Whether the absence of oxidized LDL from the
nonatheromatous areas reflects a lesser oxidative stress
or, perhaps, a sufficiency of protective activity emanating from mast cells remains to be shown.
Acknowledgment
We thank Paivi Hiironen for her excellent technical
assistance.
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Inhibition of copper-mediated oxidation of LDL by rat serosal mast cells. A novel cellular
protective mechanism involving proteolysis of the substrate under oxidative stress.
K A Lindstedt, J O Kokkonen and P T Kovanen
Arterioscler Thromb Vasc Biol. 1993;13:23-32
doi: 10.1161/01.ATV.13.1.23
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