© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Inhibition of Mast Cell–Dependent Conversion of Cultured Macrophages Into Foam Cells With Antiallergic Drugs
From the Wihuri Research Institute, Helsinki, Finland.
Correspondence to Petri T. Kovanen, Wihuri Research Institute, Kalliolinnantie 4, 00140 Helsinki, Finland. E-mail petri.kovanen{at}wri.fi
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Abstract |
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Abstract—Degranulation of isolated, rat peritoneal mast cells in the presence of low density lipoprotein (LDL) induces cholesteryl ester accumulation in cocultured macrophages with ensuing foam cell formation. This event occurs when the macrophages phagocytose LDL particles that have been bound to the heparin proteoglycans of exocytosed granules. In an attempt to inhibit such foam cell formation pharmacologically, rat peritoneal mast cells that had been passively sensitized with anti–ovalbumin-IgE were treated with 2 mast cell–stabilizing antianaphylactic drugs, MY-1250 or disodium cromoglycate (DSCG). Both drugs were found to inhibit antigen (ovalbumin)-triggered release of histamine from the mast cells, revealing mast cell stabilization. In cocultures of rat peritoneal macrophages and passively sensitized mast cells, addition of MY-1250 before addition of the antigen resulted in parallel reductions in histamine release from mast cells, uptake of [14C]sucrose-LDL, and accumulation of LDL-derived cholesteryl esters in the cocultured macrophages. Similarly, when passively sensitized mast cells were stimulated with antigen in the presence of DSCG and the preconditioned media containing all substances released from the drug-treated mast cells were collected and added to macrophages cultured in LDL-containing medium, uptake and esterification of LDL cholesterol by the macrophages were inhibited. The inhibitory effects of both drugs were mast cell–specific because neither drug inhibited the ability of macrophages to take up and esterify LDL cholesterol. Analysis of heparin proteoglycan contents of the incubation media revealed that both drugs had inhibited mast cells from expelling their granule remnants. Thus, both MY-1250 and DSCG prevent mast cells from releasing the heparin proteoglycan–containing vehicles that bind LDL and carry it into macrophages. This study suggests that antiallergic pharmacological agents could be used in animal models to prevent mast cell–dependent formation of foam cells in vivo.
Key Words: atherosclerosis • IgE antibodies • LDLs • mast cells • antiallergic drugs • macrophage foam cells
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Introduction |
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The human arterial intima contains mast cells, which often reside in the close vicinity of macrophage foam cells.1 We have previously investigated the interactions between these 2 blood-borne, immunologically active cell types in atherogenesis by asking the specific question: Could mast cells play a role in the conversion of macrophages to foam cells? Morphological studies of fatty streaks from human aortic2 and coronary3 intimas have shown that in these early lesions, the numbers of mast cells are significantly greater than in the normal intima. A series of findings by our group has delineated a sequence of events in which activation of mast cells leads to transendothelial transport of LDLs from the bloodstream to extravascular sites in rat skin4 and also enhances the uptake of LDL by mouse or rat peritoneal macrophages, with resultant foam cell formation in vitro5 6 and in vivo.7 These findings are compatible with the notion that mast cells participate in the formation of macrophage foam cells and hence, may play a role in the generation of skin xanthomas and atherosclerotic lesions.
The aforementioned experimental studies, which used mast cells stimulated either immunologically (IgE mediated) or nonimmunologically (with compound 48/80), identified a granule-remnant carrier system as a common end pathway in the mast cell–macrophage interaction that may ultimately lead to formation of foam cells.8 9 10 The cytoplasm of mast cells is filled with specific, membrane-bound organelles, the secretory granules. On stimulation of mast cells, the membranes of several individual granules fuse and a degranulation channel is formed.11 The channel membrane then fuses with the cell’s plasma membrane, pores are formed, and the channels open to the cell exterior, thereby allowing extracellular fluid (incubation medium) to enter the channels. On contact with extracellular fluid, the soluble components of granules, notably histamine and heparin proteoglycans, are detached from the granules and move from the channels into the extracellular space. The residual granules still remaining in the channels are spherical organelles with diameters ranging from 0.5 to 1 µm and are called granule remnants.8 They are composed of 2 neutral proteases (chymase and carboxypeptidase A) and heparin proteoglycans, which remain tightly bound to each other. When mast cells are stimulated vigorously, some of their granule remnants are expelled from the degranulation channels into the vicinity of the parent cells. Within 10 minutes of stimulation, the degranulation channels close, and the mast cells start to reconstitute the granules from the remnants by replenishing the lost constituents.
When isolated rat serosal mast cells are stimulated with compound 48/80 in the presence of LDL, the LDL becomes bound to the granule remnants present in the degranulation channels and to those expelled from the channels.10 The expelled granule remnants with their LDL load are then phagocytosed by cocultured macrophages, with the ensuing formation of macrophage foam cells. In addition, LDL binds to the soluble heparin proteoglycans and forms insoluble complexes with them. These insoluble complexes are also phagocytosed by cocultured macrophages.12
Like 48/80-triggered mast cell stimulation, IgE-dependent stimulation of sensitized mast cells also leads to increased uptake of LDL by cocultured macrophages and foam cell formation.6 IgE-mediated release of histamine from mast cells is calcium dependent,13 and antiallergic drugs such as disodium cromoglycate (DSCG) and MY-1250, which is an active metabolite of the new, orally active, antiallergic drug repirinast, exert their antisecretory effect by inhibiting the influx of extracellular Ca2+.14 15
In the present study, we used a system in which rat peritoneal mast cells and macrophages were cocultured in the presence of LDL. Before incubation, the mast cells were sensitized by allowing them to bind anti–ovalbumin-IgE. To stabilize the mast cells and prevent their immunological activation by the specific antigen (ovalbumin), we used MY-1250 or DSCG. The results show that this pharmacological inhibition of mast cell degranulation blocks mast cell–dependent LDL uptake and the ensuing accumulation of cholesteryl esters by cocultured macrophages. This inhibitory effect of the 2 drugs on foam cell formation was mast cell–specific, suggesting that they can be used in vivo to test the hypothesis that mast cells participate in the formation of foam cells.
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Methods |
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Animals, Reagents, and Materials
Male Wistar rats 9 to 20 weeks old and weighing 200 to 350 g were used throughout the study. The rats were obtained from the Laboratory Animal Center of the University of Helsinki, Helsinki, Finland. Ovalbumin, heparin, substance P, compound 48/80, and bovine serum albumin (BSA) were from Sigma; aluminum hydroxide gel from Merck; RPMI 1640 culture medium supplemented with 25 mmol/L HEPES and Dulbecco’s phosphate-buffered saline (PBS) from Gibco; Eagle’s basal medium with Earle’s salts (EBME) with 20 mmol/L HEPES from Flow Laboratories; mouse monoclonal antibody to rat IgE heavy chain (MARE-1), standard rat IgE myeloma IR162, and horseradish peroxidase–conjugated mouse monoclonal antibody to rat
light chain (MARK-1; MCA 187p) from Serotec;
3',3',5',5',-tetramethylbenzidine from Kirkegaard & Perry Laboratories
Inc; and
[U-14C]sucrose
(200 Ci/mol) and [1-14C]oleic acid (53
mCi/mmol) from Amersham International. Alcian Blue 8 GS was from Fluka,
Bond Elute columns (NH2-bonded silica) were from
Varian, and plastic dishes were from Falcon. Pertussis vaccine was
obtained from the National Public Health Institute of Finland. DSCG
(Lomudal, 10 mg/mL) was from Fisons Pharmaceuticals. MY-1250 was a kind
gift from the Mitsubishi Chemical Corporation, Yokohama,
Japan.
Preparation of IgE Serum
IgE-containing immune serum was obtained from rats 3
to 4 weeks after immunization with ovalbumin as the antigen, as
described previously.6 The
concentration of IgE as measured by
ELISA16 was 38 µg/mL in
the pooled (from 20 rats) immune serum. The IgE-containing serum was
stored in aliquots at -70°C, thawed once, and used for the
experiments.
Passive Sensitization of Peritoneal Mast
Cells
For passive sensitization of mast cells, peritoneal
cells were obtained from nonimmunized rats by peritoneal lavage,
essentially as described previously for isolation of peritoneal cells
from normal rats.17 The
peritoneal cells (mast cells and macrophages) were resuspended
with 50% (vol/vol) of IgE-containing serum in PBS (7 to
10x105 mast cells per mL) and incubated in
50-mL Falcon tubes at 37°C for 3 hours to allow the IgE to bind to
the mast cells. After incubation, the IgE-containing serum was removed
by centrifuging the cells twice in PBS, and finally the sensitized mast
cells were resuspended in culture medium, transferred to culture
dishes, and used for experiments.
Isolation of LDL and Preparation of
[14C]Sucrose-LDL, Colloidal Gold–LDL
Conjugates, and Acetyl-LDL
Human LDL
(d=1.019 to 1.050 g/mL) was
isolated from plasma by sequential ultracentrifugation
in the presence of 3 mmol/L Na2EDTA, as
described.18 The isolated
LDL was labeled with [14C]sucrose to yield
specific activities in the range of 20 to 30 disintegrations per minute
per nanogram of LDL
protein.19 The concentration
of the LDL preparations is expressed in terms of their protein
concentration. Colloidal gold particles (15 nm) were prepared and
conjugated with LDL, as described by Robenek et
al.20 Gold-LDL preparations
were examined by transmission electron microscopy by using negative
staining and were found to contain 3 to 5 LDL molecules per gold
particle. The concentrations of the gold-LDL preparations are expressed
in terms of their protein concentration. Acetyl-LDL was prepared by
treatment of LDL with acetic
hydride.21
Effect of MY-1250 and DSCG on Histamine Release
From Passively Sensitized Mast Cells
Passively sensitized mast cells
(5x104) were preincubated at 37°C for 5
minutes in 200 µL of PBS. MY-1250 or DSCG was then added to give the
indicated concentrations, and after preincubation for 20 seconds, a
mast cell–degranulating agent (ovalbumin, substance P, or
compound 48/80) was added and incubation was continued for 10 minutes
to allow completion of mast cell degranulation. The reaction was
stopped by immersing the tubes in ice-cold water, and the mast cells
were sedimented by centrifugation at
400g for 5 minutes at 4°C.
The histamine contents of both supernatant and sediment were determined
fluorometrically according to Bergendorff and
Uvnäs,22 with
modifications.23 The degree
of histamine release, ie, the amount of histamine in the supernatant,
was expressed as a percentage of the total histamine (ie, the sum of
histamine contents in the supernatant and
sediment).
Electron Microscopy of Peritoneal Cell
Cultures
After incubation of peritoneal cells with mast
cell–degranulating and/or –stabilizing agents, the cells were
sedimented and the pellets thus formed were fixed in situ with 2%
glutaraldehyde at room temperature for 1 hour and
dehydrated; the contents of the tubes were mounted in Epon 112
embedding medium. After 2 days (to allow for polymerization), the Epon
blocks were sectioned horizontally along the cell layers.
Ultramicrotome sections (60 nm) were stained with uranyl acetate and
lead citrate and viewed and photographed in a JEOL 1200 EX electron
microscope at 60 kV in the Department of Electron Microscopy,
University of Helsinki, Helsinki, Finland.
To visualize uptake of gold-LDL by macrophages, actively sensitized mast cells were cocultured with macrophages and stimulated with ovalbumin. Then, 30 µg/mL gold-LDL was added and the incubation was continued for 4 hours. After incubation, the macrophage monolayers were fixed in their culture dishes with 2% glutaraldehyde at room temperature for 1 hour and dehydrated, and the contents of the dishes were mounted in Epon 112 embedding medium and prepared for electron microscopy.
Effect of MY-1250 on IgE-Mediated Uptake of
[14C]Sucrose-LDL or Unlabeled LDL by
Macrophages
Passively sensitized mast cells
(105 cells per well) and macrophages
(106 cells per well) were incubated in 800
µL of EBME containing 10 mg/mL BSA and 100 IU/mL penicillin (medium
A) in a 5% CO2 atmosphere for 1 hour at 37°C.
After a 20-second pretreatment of mast cells with MY-1250 at the
concentrations indicated (0 to 200 µg/mL), 10 µg/mL
ovalbumin was added and incubation was continued for 30
minutes. Finally, 15 to 80 µg of
[14C]sucrose-LDL was added and incubation
was continued for the periods indicated. When incubation ended, the
media were removed. and the macrophage monolayers were washed
twice with 1 mL of PBS containing 10 mg/mL BSA and 10 mg/mL heparin and
then washed twice more with 1 mL of PBS. Histamine was determined from
a 200-µL sample of the medium from which the cells had been removed
by centrifugation. The macrophage monolayers
were dissolved in 0.2N NaOH, and their contents of
14C radioactivity and DNA were determined.
To study the effect of MY-1250 on mast cell–dependent uptake of LDL by
macrophages, mast cells and macrophages were cocultured
with 100 µg/mL native LDL for 38 hours under the same conditions as
above. After incubation, the cholesteryl esters in the
macrophages were measured.
Effect of DSCG Treatment of Mast Cells on Mast
Cell–Dependent Incorporation of
[14C]Oleate Into Cholesteryl Esters by
Macrophages
Passively sensitized mast cells
(4.5x106) were incubated in 1 mL of PBS at
37°C, and 15 µL of DSCG was added to the cells to give a final
concentration of 300 µmol/L. As a control, passively sensitized mast
cells to which 15 µL of PBS had been added were used. Immediately
after addition of DSCG or PBS, the cells were challenged with
500 µg/mL ovalbumin, and incubation was continued for a
further 30 minutes at 37°C. The cells were then sedimented by
centrifugation in the cold, and the supernatants were
collected ("preconditioned medium"). Finally, 130 µL of the
preconditioned medium corresponding to
8x105 passively sensitized mast cells was
added to the macrophage monolayers
(3x106 macrophages per well). Each
macrophage well received 1 mL of medium B containing 50 µg of
LDL and 20 µmol/L
[14C]oleate-albumin (123 000
dpm/nmol). After incubation at 37°C for 20 hours, the cells were
washed, their lipids were extracted with hexane/isopropyl alcohol (3:2,
vol/vol), and the cellular content of cholesteryl
[14C]oleate was determined by thin-layer
chromatography.23
Measurement of Cholesteryl Esters in
Macrophage Monolayers
After incubation, the macrophage monolayers
were washed and the cell lipids were extracted with hexane/isopropanol
(3:2, vol/vol) and applied to Bond Elute columns
(NH2-bonded silica) to separate the cholesteryl
esters from the fatty acids and phospholipids. The quantities of
cholesteryl esters (oleate and linoleate) were then determined by
high-performance liquid
chromatography.24
Measurement of Cellular DNA
After incubation, the cells in each well were washed
and dissolved in 0.5 mL of a mixture containing 0.2% Triton X-100 and
1 mmol/L NaOH, and cellular DNA was
assayed.25 Calf thymus DNA
was used as a standard.
Other Assays
Protein was determined by the procedure of Lowry et
al26 with BSA as the
standard. 14C radioactivity was measured in
a liquid scintillation counter (1215 Rackbeta) and expressed as dpm.
The amount of Alcian Blue–reactive material released from the
stimulated mast cells was measured by the method of Bartold and
Page.27
Statistical Analysis
The results were processed by SigmaPlot. Descriptive
statistics are presented as mean values of the experiments and
their SEMs. Differences between groups were tested by using Student’s
t test and were considered
significant when
P<0.05.
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Results |
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Rat peritoneal mast cells were passively sensitized by incubating them with immune serum containing high titers of anti–ovalbumin-IgE. MY-1250 or DSCG was then added to the IgE-bearing mast cells in quantities to give the concentrations indicated (Figures
1A and 1B). In the absence of drugs, 
40% of the
cellular histamine was released, as a sign of the massive degranulation
in response to the immunological stimulus. The histamine release was
inhibited strongly by both MY-1250 and DSCG in a dose-dependent
fashion, the near-maximal effect being observed at 5 µg/mL and 5
µmol/L for MY-1250 and DSCG, respectively.
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Using drug concentrations that strongly inhibited histamine
release (100 µg/mL MY-1250 and 30 µmol/L DSCG), we then tested the
2 drugs for their ability to inhibit the release of heparin
proteoglycans from IgE-bearing mast cells challenged with
ovalbumin. As shown in
Figure
2, in the absence of drugs, ovalbumin
triggered the release of both histamine and heparin proteoglycans (A
versus B; 19-fold and 5-fold increase, respectively), the
ovalbumin-triggered release of heparin proteoglycans (16%)
being less than that of histamine (38%). This finding can be explained
by the fact that histamine is also released from the heparin-containing
granules that remain in the degranulation channels of stimulated mast
cells. However, both drugs did inhibit the release of heparin
proteoglycans, as they did the release of histamine (C and D).
In additional experiments, we measured the content of insoluble heparin
proteoglycans (ie, of granule remnants) and of soluble heparin
proteoglycans in mast cell releasates and found that release of both
insoluble and soluble heparin proteoglycans was inhibited by either
drug (not shown).
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We then performed a series of experiments with the drug
MY-1250 to investigate whether pharmacological stabilization of mast
cells could inhibit mast cell–mediated uptake of LDL by cocultured
macrophages. First, the ability of this drug to inhibit
degranulation of mast cells stimulated in various ways was studied. In
the experiment shown in
Figure
3A, the cells were challenged with
ovalbumin, substance P, or compound 48/80, a specific,
noncytotoxic stimulator of mast cells. As expected, when passively
sensitized mast cells were stimulated immunologically with
ovalbumin, inhibition of histamine release from the mast cells
was dose dependent. In contrast, no inhibition was observed when the
cells were stimulated nonimmunologically with either substance P or
compound 48/80. We then tested the effect of MY-1250 on the ability of
anti-IgE to stimulate IgE-bearing mast cells
(Figure
3B). Again, we observed a dose-dependent inhibition
by MY-1250 of immunologically induced histamine release from the
passively sensitized mast cells. Nonsensitized mast cells served as
controls. Here the mast cell–stimulating effect of anti-IgE was
minimal, probably reflecting the presence of nonspecific IgE molecules
on the mast cells (addition of the specific antigen ovalbumin
had no stimulating effect). However, MY-1250 also tended to inhibit
this nonspecific immunological challenge to mast cells.
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Based on the above results, a series of experiments with
MY-1250 was performed by using anti–ovalbumin-IgE–bearing
mast cells. First, for morphological observations, the cells obtained
by peritoneal lavage (mast cells and macrophages) were fixed
and studied by electron microscopy. Untreated rat peritoneal mast cells
had a typical appearance; ie, they were filled with electron-dense
granules
(Figure
4A). After passive sensitization with
anti–ovalbumin-IgE and a challenge with ovalbumin,
most of the mast cells showed signs of massive degranulation, as
revealed by the appearance of electron-lucent granule remnants both
intracellularly (in large intracellular cavities or degranulation
channels) and extracellularly (around the mast cells;
Figure
4B). On exposure to the extracellular fluid, the
granules become electron-lucent when they released their histamine and
a fraction of their heparin proteoglycans in soluble form (not
visible). Interestingly, the cocultured macrophages contained
phagocytosed granule remnants as soon as 10 minutes after stimulation
of the mast cells
(Figure
4B). When the cocultures were treated with MY-1250
before the ovalbumin challenge, signs of secretory activity,
ie, granule exocytosis, were seen only sporadically
(Figure
4C). Indeed, in their ultrastructure, mast cells
treated with MY-1250 closely resembled unstimulated mast cells (see
Figure
4A).
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Mast cell activation with ensuing degranulation leads to the
formation of 2 extracellular pools of heparin proteoglycans: the
"solid-phase heparin proteoglycans" present in the granule
remnants and the "soluble heparin proteoglycans" released from the
granules when the remnants are formed. LDL binds to both pools of
heparin proteoglycans, and this binding can be visualized by
transmission electron microscopy if the LDL particles have been labeled
with colloidal
gold.6 12 The
LDL-coated granule remnants and insoluble complexes formed between LDL
and the soluble heparin proteoglycans are then phagocytosed by
specialized phagocytes, such as macrophages. Steps of the 2
parallel pathways of mast cell–dependent LDL uptake by cultured
macrophages are visualized in
Figure
5. In this photograph, both extracellular and
intracellular (within a macrophage) LDL-coated granule remnants
(asterisks) and insoluble LDL–heparin proteoglycan aggregates (arrows)
are visible.
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To quantify the ability of MY-1250 to inhibit mast
cell–dependent uptake of LDL by macrophages, we used
[14C]sucrose-labeled LDL because sucrose
becomes trapped within lysosomes, and it is therefore possible
to measure the cumulative uptake of the labeled
ligands.19 Mast cells and
macrophages obtained from the peritoneal cavities of untreated
rats were preincubated in serum with a high concentration of
anti–ovalbumin-IgE to sensitize the mast cells. After
sensitization, cell aliquots were treated with MY-1250 in increasing
concentrations, and [14C]sucrose-LDL and
ovalbumin were then added to the incubation medium. As shown in
Figure
6, dose-dependent inhibition of IgE-mediated
[14C]sucrose-LDL uptake by the cocultured
macrophages ensued when MY-1250 was present in the
incubation medium. This reduction of LDL uptake by macrophages
closely followed the inhibition of histamine release by the mast cells
(inset).
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The phagocytosed, granule remnant–bound LDL particles are
rapidly degraded by lysosomes of the macrophages, and
simultaneously the rate of cholesteryl ester synthesis in
these cells increases,5 which
suggests that cholesteryl esters should accumulate in the cytoplasm. To
study the effect of MY-1250 on the accumulation of cholesteryl esters
in macrophages, we cocultured macrophages with
antigen-stimulated mast cells for 38 hours in LDL-containing medium in
the absence or presence of MY-1250. As shown in
Figure
7, LDL and ovalbumin alone had little
effect on macrophage cholesteryl ester content (column A).
However, when mast cells were present and ovalbumin was
absent (B), the cholesteryl ester content rose by 3-fold, owing to
the spontaneous degranulation of mast cells during the 38-hour
incubation. Stimulation of mast cells with ovalbumin further
increased the cholesteryl ester content of the macrophages (C).
Stabilization of mast cells with MY-1250 fully prevented the
ovalbumin-dependent increase in cholesteryl esters in the
cocultured macrophages (D).
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We then investigated whether the classic mast
cell–stabilizing agent DSCG would also inhibit the mast cell–induced
uptake of LDL. In the experiment shown in
Figure
8, instead of coculturing the mast cells with
macrophages, we stimulated the mast cells for 30 minutes,
collected the released material, and then added it to the
macrophage cultures. This procedure was done to minimize the
effect of spontaneous degranulation of mast cells, which occurs during
long coculture incubations. (Results obtained with the 2 antiallergic
drugs were essentially the same, whether intact mast cells or mast cell
releasate was added to the macrophage cultures.). Sensitized
mast cells (4.5x106 cells) were stimulated
immunologically with ovalbumin in the presence or absence of
DSCG, and the cells were removed by sedimentation to obtain
preconditioned media of 2 kinds. The preconditioned media were then
added to macrophages cultured in the presence of LDL, and
incorporation of [14C]oleate into
cholesteryl [14C]oleate in the
macrophages for 20 hours was measured as an index of LDL uptake
and cholesterol
esterification.23 The
preconditioned medium from mast cells that had been stimulated in the
absence of DSCG significantly increased the rate of cholesteryl ester
formation in the macrophages
(Figure
8; B versus A). In contrast, when the
preconditioned medium from DSCG-treated mast cells was added to the
macrophages, no increase in cholesterol
esterification was observed (C). Thus, mast cell stabilization by
either drug resulted in loss of the ability of mast cells to induce
uptake of LDL by cultured macrophages.
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Finally, we tested for the cell specificity of the 2 antiallergic drugs. Neither drug, when added to give the concentrations used in the above experiments, had an effect on the ability of cultured macrophages to incorporate acetyl-LDL–derived cholesterol into cytoplasmic cholesteryl esters (not shown). Thus, the effect of the 2 drugs on mast cell–dependent uptake of LDL by macrophages must have been mast cell–specific.
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Discussion |
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The present results demonstrate that antiallergic drugs with a mast cell–stabilizing effect can inhibit mast cell–dependent foam cell formation in vitro. The inhibitory effect depended on stabilization of immunologically sensitized mast cells. This stabilization was associated with decreased exocytosis of heparin proteoglycans from the mast cells. Analysis of the proteoglycans present in the mast cell releasates revealed that the 2 mast cell–stabilizing drugs, MY-1250 and DSCG, had inhibited the mast cells from expelling their heparin proteoglycan–containing granule remnants and also inhibited them from releasing soluble heparin proteoglycans. These inhibitory effects of the partial stabilization of mast cells on the release of the 2 types of heparin proteoglycans (solid-phase and soluble) have not been fully recognized previously. The current results provide a mechanistically plausible explanation for the strong, inhibitory effect of the antiallergic drugs on LDL uptake by macrophages.
Some of the antiallergic drugs originally designed as mast cell–stabilizing drugs, notably tranilast, not only are mast cell–specific but also influence the metabolism of other cells, such as smooth muscle cells.28 Similarly, DSCG has been suggested to affect other cells besides mast cells.29 30 Therefore, it was important to examine whether the 2 drugs that we used had any direct effect on macrophage LDL metabolism. However, the rates of LDL uptake and subsequent cholesterol esterification by macrophages in the absence and presence of the drugs were the same. Accordingly, in this simple coculture system, pharmacological inhibition of mast cell–dependent foam cell formation could be attributed solely to inhibition of the release of the 2 forms of heparin proteoglycans from the mast cells, which thus partially or totally blocked the 2 parallel heparin proteoglycan–dependent pathways for carrying LDL into the macrophages. Clearly, the effects of these drugs on foam cell formation will need to be reexamined in multicellular systems and eventually in animal models in vivo.
Any stimulus causing mast cells to degranulate would be sufficient to initiate a sequence of events that might ultimately lead to formation of foam cells. What, then, are the actual stimuli in vivo that could lead to mast cell–dependent foam cell formation, and how could they be prevented pharmacologically? There are 2 clinically relevant sites in which foam cells are formed and in which mast cells are also present: the skin, where xanthomas form, and the arterial intima, where fatty streaks and atheromas form. In the skin, in addition to the classic IgE-mediated mast cell degranulation, neural stimulation of mast cells due to release of substance P has been demonstrated.31 Furthermore, mast cells in the skin can be stimulated by mechanical strain (vibration).32 Regarding the atherosclerotic arterial intima, the actual stimulators of mast cells have not been studied. However, atherosclerotic lesions appear to contain a wide variety of agents potentially capable of triggering mast cells to extrude their granules. These include activated complement33 34 and 2 types of activated inflammatory cells, T cells35 and macrophages,36 both of which may liberate histamine-releasing factors capable of stimulating mast cells. Thus, mast cells of the atherosclerotic arterial intima reside in an immunologically active area, where they are locally exposed to many potential stimuli and so become activated. Interestingly, arterial mast cells are responsive to compound 48/80, revealing a similarity to the rat serosal mast cells used in this study.37 The responsiveness of mast cells to different stimulators also varies; eg, in contrast to the rat serosal mast cells used in this study, lung mast cells are unresponsive to compound 48/80.38
Differences also exist among mast cell
stabilizers. Whereas compound MY-1250 inhibits IgE-mediated stimulation
of mast cells (see
Figure
3), other antiallergic agents such as DSCG,
ketotifen, and tranilast also inhibit substance P–induced histamine
release from mast cells.39
Moreover, DSCG inhibits 48/80-mediated stimulation of rat serosal mast
cells. Clearly, functional and pharmacological differences exist among
mast cells in various anatomic sites. Therefore, studies are needed to
unravel the actual mast cell stimulators in the arterial
wall (and skin) and to determine which antiallergic drugs can reach
these target tissues and effectively inhibit the action of
tissue-specific mast cell–degranulating agents.
The mast cell–stabilizing drugs, by preventing mast cell stimulation, block release not only of proteoglycans but also of various vasoactive compounds that are released on activation of mast cells. The vasoactive compounds, notably histamine, dramatically increase the transendothelial flux of LDL particles into rat skin in vivo.4 In the skin, as in other extrahepatic tissues, the concentration of LDL is much lower (only 1/10) than in the corresponding blood plasma.40 Thus, in the skin, mast cell activation leads to a rapid increase of LDL concentration in the extracellular fluid and so creates conditions suitable for the binding of LDL to exocytosed heparin proteoglycans.4 By blocking this increase, a mast cell–stabilizing drug would have an additional benefit in preventing mast cell–dependent foam cell formation in the skin. In the arterial intima, in contrast to extrahepatic tissues, the concentration of LDL appears to equal that in the corresponding plasma,41 and accordingly, no such acute and dramatic effect of mast cell histamine or other vasoactive compounds on LDL concentration would be expected. However, because accumulation of lipoprotein-derived lipids in the arterial intima is a slow and long-lasting process, even subtle increases in the influx of lipoproteins could result in the early appearance of atherosclerotic lesions. Finally, stimulation of rat serosal mast cells leads to release of proteolytic enzymes, notably chymase and carboxypeptidase A.42 These 2 enzymes are tightly bound to the heparin proteoglycans of granule remnants, and, when degrading the apoB-100 component of granule remnant–bound LDL particles, they act in concert. The proteolyzed remnant-bound particles then become unstable and fuse into larger lipid droplets, which remain bound to the remnants with even higher affinity than do native LDL particles.43 In this way, the average capacity of an exocytosed granule to bind and carry LDL is greatly increased.44 Because pharmacological treatment of mast cells with mast cell stabilizers blocks the release of proteases, the protease-dependent stimulation of mast cell–dependent foam cell formation is also inhibited.
In summary, mast cells appear to have multiple regulatory effects on the metabolism of LDL particles along their route from the circulation to the macrophages. These effects are produced by various compounds that are released from stimulated mast cells; at least 20 chemical mediators are released from mast cells after their stimulation.45 The actions of these mediators on LDL metabolism could be inhibited individually by pharmacological means, eg, the action of histamine by the use of antagonists of histamine receptors, the action of leukotrienes by leukotriene receptor antagonists, the action of chymase by the use of specific chymase inhibitors, and the action of heparin proteoglycans by protamine sulfate. Considering the multitude of actions that stimulated mast cells have on LDL metabolism, it appears that the ideal candidate for in vivo testing of the inhibition of mast cell–mediated foam cell formation in experimental animals would be a specific mast cell stabilizer, which, through its inherent mode of action, would be capable of blocking all of the effects that activated mast cells may have on LDL metabolism. This report provides the first rationale for such pharmacological tests.
Received April 26, 2000; accepted September 21, 2000.
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