Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents
Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:e134-e142

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ma, H.
Right arrow Articles by Kovanen, P. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ma, H.
Right arrow Articles by Kovanen, P. T.
Related Collections
Right arrow Pathophysiology
Right arrow Lipid and lipoprotein metabolism
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:e134.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Inhibition of Mast Cell–Dependent Conversion of Cultured Macrophages Into Foam Cells With Antiallergic Drugs

Hua Ma; Petri T. Kovanen

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


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {kappa} 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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 (FiguresDown 1A and 1B). In the absence of drugs, {approx}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.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. Concentration-dependent inhibition by MY-1250 and DSCG on histamine release from passively sensitized mast cells after immunological stimulation. Mast cells were passively sensitized with rat immune serum containing a high concentration of anti–ovalbumin-IgE antibodies, as described in Methods. The passively sensitized mast cells (5x104) were then treated with the indicated concentrations of MY-1250 or DSCG. Immediately after treatment, the cells were stimulated immunologically with ovalbumin (10 µg/mL), and incubation was continued for 15 minutes to allow completion of mast cell degranulation. The reactions were stopped by sedimenting the cells in the cold, and the histamine content of both supernatant and sediment was determined fluorometrically. The percent histamine release was calculated as described in Methods and was taken to reflect the degree of mast cell degranulation. Values are mean±SEM (n=3).

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 FigureDown 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).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Inhibitory effect of MY-1250 and DSCG on the release of histamine and heparin proteoglycans from passively sensitized mast cells after immunological stimulation. Mast cells were passively sensitized with rat immune serum containing a high concentration of anti–ovalbumin-IgE antibodies, as described in Methods. The passively sensitized mast cells (5x104) were then pretreated with buffer (A and B), MY-1250 (100 µmg/mL, C), or DSCG (30 µmol/L, D). After pretreatment, either the cells were left unstimulated (A) or they were stimulated immunologically with ovalbumin (10 µg/mL), and incubation was continued for 15 minutes to allow completion of mast cell degranulation (B, C, and D). Note that control mast cells did not receive any of the above agents. The reactions were stopped by sedimenting the cells in the cold, and the content of histamine and heparin proteoglycans in both supernatant and sediment was determined. The percent values for histamine and heparin proteoglycan release were calculated as described in Methods. Values are mean±SEM (n=3). The inhibitory effects of MY-1250 and DSCG on both histamine and heparin proteoglycan release were statistically significant (P<0.005).

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 FigureDown 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 (FigureDown 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.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Effect of MY-1250 on nonimmunological and immunological stimulation of passively sensitized mast cells. Mast cells were passively sensitized with rat immune serum containing a high concentration of anti–ovalbumin-IgE antibodies, as described in Methods. The passively sensitized mast cells (5x104) were then treated with the indicated concentrations of MY-1250 for 20 seconds. After pretreatment, the cells were stimulated nonimmunologically with compound 48/80 (1 µg/mL) or substance P (10–4 mol/L) or immunologically with ovalbumin (10 µg/mL; A), and incubation was continued for 10 minutes to allow completion of mast cell degranulation. In B, the experimental design was identical, except that anti-IgE was added as an immunological stimulant and nonsensitized mast cells were used as controls. The reactions were stopped by sedimenting the cells in the cold. The histamine content of both supernatant and sediment was determined fluorometrically. The degree of histamine release was calculated as described in Methods. Similar results were obtained in 2 other independent experiments.

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 (FigureDown 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; FigureDown 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 (FigureDown 4B). When the cocultures were treated with MY-1250 before the ovalbumin challenge, signs of secretory activity, ie, granule exocytosis, were seen only sporadically (FigureDown 4C). Indeed, in their ultrastructure, mast cells treated with MY-1250 closely resembled unstimulated mast cells (see FigureDown 4A).



View larger version (78K):
[in this window]
[in a new window]
  		Figure 4. Effect of MY-1250 on morphology of immunologically stimulated mast cells. Peritoneal cells (mast cells and macrophages) were obtained from nonimmunized rats by peritoneal lavage, sedimented, and fixed for electron microscopy (A). Another sample of peritoneal cells was then incubated with anti–ovalbumin-IgE–containing serum to passively sensitize the mast cells. Finally, the cells were incubated for 20 seconds in either the absence (B) or presence (C) of 200 µg/mL MY-1250 and immediately thereafter challenged with 10 µg/mL ovalbumin. Incubation was continued for 10 minutes to allow completion of mast cell degranulation. The reaction was stopped by sedimenting the cells in the cold, and the pellets were then fixed for electron microscopy. Note extensive degranulation in the absence of MY-1250 and minimal degranulation in its presence. MC indicates mast cell; MØ, macrophage; EOS, eosinophil; and *, granule remnant. Original magnification x2000 in A and B, x3000 in C; bar=2 µm.

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 FigureDown 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.



View larger version (130K):
[in this window]
[in a new window]
  		Figure 5. Photomicrograph showing uptake of granule remnant–bound gold-LDL and of heparin proteoglycan–gold-LDL aggregates (insoluble complexes) by macrophages. Peritoneal cells (mast cells and macrophages) were obtained from nonimmunized rats by peritoneal lavage and cocultured for 1 hour. The mast cells were passively sensitized by incubation with anti–ovalbumin-IgE–containing serum. The mast cells were then stimulated with ovalbumin, and incubation was continued for 10 minutes to allow completion of mast cell degranulation and closure of degranulation channels. Finally, gold-LDL (30 µg/mL) was added, and incubation was continued for 4 hours. After incubation, the medium was removed and the monolayer containing both mast cells and macrophages was fixed for electron microscopy. Note both extracellular and intracellular gold-LDL–coated granule remnants and aggregates of gold-LDL (insoluble complexes with heparin proteoglycans). In the aggregates, only colloidal gold (black dots) is visible. MC indicates mast cell; MØ, macrophage; *, granule remnant; and arrow, heparin proteoglycan–gold-LDL complexes (aggregate). Original magnification x5000; bar=1 µm.

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 FigureDown 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).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 6. MY-1250 inhibits mast cell–dependent uptake of [14C]sucrose-LDL by macrophages. Macrophages (106 cells) and passively sensitized mast cells (105) were cocultured in medium A for 1 hour at 37°C. The cells were first treated with the indicated concentrations of MY-1250 for 20 seconds and then challenged with 10 µg/mL ovalbumin for 30 minutes. Finally, 60 µg of [14C]sucrose-LDL was added, and incubation was continued for 15 hours. The media were then collected and their histamine concentrations determined. Macrophage monolayers were washed and dissolved in 0.2N NaOH, and their contents of 14C radioactivity and DNA were determined. Uptake of LDL by macrophages was determined as described in Methods. Values are mean±SEM (n=3).

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 FigureDown 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 {approx}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).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 7. MY-1250 inhibits accumulation of LDL-derived cholesteryl esters in macrophages cocultured with immunologically activated mast cells. Macrophages (106 cells) and passively sensitized mast cells (105) were cocultured in medium for 2 hours at 37°C. In 1 set of dishes (A), the mast cells were removed by washing the macrophage monolayers, and fresh medium A was added. In other dishes (B, C, and D), the mast cells were present throughout the experiment. To the cells in D, MY-1250 was added to give a final concentration of 100 µg/mL. Each group of cells (except B) was then challenged with 10 µg/mL ovalbumin for 30 minutes, LDL was added to give a final concentration of 100 µg/mL, and incubation was continued for 38 hours at 37°C. The media were then removed, the macrophage monolayers were washed, their lipids were extracted, and the quantities of cholesteryl esters (oleate and linoleate) were determined by high-performance liquid chromatography. Values are mean±SEM (n=4). For A vs B, B vs C, and C vs D, all differences were statistically significant (P<0.005).

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 FigureDown 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 (FigureDown 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.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 8. DSCG treatment of mast cells inhibits 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 and challenged with ovalbumin in either the absence or presence of DSCG (300 µmol/L), and aliquots of preconditioned media corresponding to 8x105 mast cells were added to macrophage monolayers (3x106 macrophages per well), which resulted in a 1:5 dilution of DSCG (concentration in macrophage medium was 60 µmol/L). Control macrophages did not receive preconditioned medium (A). Macrophages in B and C received preconditioned media derived from mast cells that had been challenged without or with prior DSCG treatment, respectively. Each macrophage well received 1 mL of medium B containing 50 µg LDL and 20 µmol/L [14C]oleate-albumin (123 000 dpm/nmol). After incubation at 37°C for 20 hours, the cellular content of cholesteryl [14C]oleate was determined. Values are mean±SEM (n=5).

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.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 FigureUp 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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Takebayashi S, Kubota I, Kamio A, Takagi T. Ultrastructural aspects of human atherosclerosis: role of the foam cells and modified smooth muscle cells. J Electron Microsc. 1972;21:301–313.[Abstract/Free Full Text]
  2. Kaartinen M, Penttilä A, Kovanen PT. Mast cells of two types differing in neutral protease composition in the human aortic intima: demonstration of tryptase- and tryptase/chymase-containing mast cells in normal intimas, fatty streaks, and the shoulder region of atheromas. Arterioscler Thromb. 1994;14:966–972.[Abstract/Free Full Text]
  3. Kaartinen M, Penttilä A, Kovanen PT. Accumulation of activated mast cells in the shoulder region of human coronary atheroma, the predilection site of atheromatous rupture. Circulation. 1994;90:1669–1678.[Abstract/Free Full Text]
  4. Ma H, Kovanen PT. Degranulation of cutaneous mast cells induces transendothelial transport and local accumulation of plasma LDL in rat skin in vivo. J Lipid Res. 1997;38:1877–1887.[Abstract]
  5. Kokkonen JO, Kovanen PT. Stimulation of mast cells leads to cholesterol accumulation in macrophages in vitro by a mast cell granule-mediated uptake of low density lipoprotein. Proc Natl Acad Sci U S A. 1987;84:2287–2291.[Abstract/Free Full Text]
  6. Ma H, Kovanen PT. IgE-dependent generation of foam cells: an immune mechanism involving degranulation of sensitized mast cells with resultant uptake of LDL by macrophages. Arterioscler Thromb Vasc Biol. 1995;15:811–819.[Abstract/Free Full Text]
  7. Kokkonen JO. Stimulation of rat peritoneal mast cells enhances uptake of low density lipoproteins by rat peritoneal macrophages in vivo. Atherosclerosis. 1989;79:213–223.[Medline] [Order article via Infotrieve]
  8. Kovanen PT. Mast cell granule-mediated uptake of low density lipoproteins by macrophages: a novel carrier mechanism leading to the formation of foam cells. Ann Med. 1991;23:551–559.[Medline] [Order article via Infotrieve]
  9. Kovanen PT. The mast cell: a potential link between inflammation and cellular cholesterol deposition in atherogenesis. Eur Heart J. 1993;14(suppl K):105–117.
  10. Kovanen PT. Role of mast cells in atherosclerosis. Chem Immunol. 1995;62:132–170.[Medline] [Order article via Infotrieve]
  11. Röchlich P, Anderson P, Uvnäs B. Electron microscope observations on compound 48/80-induced degranulation in rat mast cells: evidence for sequential exocytosis of storage granules. J Cell Biol. 1971;51:465–483.
  12. Lindstedt KA, Kokkonen JO, Kovanen PT. Soluble heparin proteoglycans released from stimulated mast cells induce uptake of low density lipoproteins by macrophages via scavenger receptor-mediated phagocytosis. J Lipid Res. 1992;33:65–75.[Abstract]
  13. Foreman JC, Hallett MB, Mongar JL. The relationship between histamine secretion and 45calcium uptake by mast cells. J Physiol. 1977;271:193–214.[Abstract/Free Full Text]
  14. Foreman JC, Garland LG. Cromoglycate and other antiallergic drugs: a possible mechanism of action. BMJ. 1976;1:820–821.
  15. Takei M, Endo K, Takahashi K. Mechanism of action of MY-1250, an active metabolite of repirinast, in inhibiting histamine release from rat mast cells. Br J Pharmacol. 1992;105:587–590.[Medline] [Order article via Infotrieve]
  16. Holmes BJ, Diaz-Sanchez D, Lawrence RA, Bell EB, Maizels RM, Kemeny DM. The contrasting effects of CD8+ T cells on primary, established and Nippostrongylus brasiliensis-induced IgE responses. Immunology. 1996;88:252–260.[Medline] [Order article via Infotrieve]
  17. Kokkonen JO, Kovanen PT. Accumulation of low density lipoproteins in stimulated rat serosal mast cells during recovery from degranulation. J Lipid Res. 1989;30:1341–1348.[Abstract]
  18. Kokkonen JO, Kovanen PT. Low density lipoprotein degranulation by rat mast cells: demonstration of extracellular proteolysis caused by mast cell granules. J Biol Chem. 1985;260:14756–14763.[Abstract/Free Full Text]
  19. Pittman RC, Green SR, Attie AD, Steinberg D. Radiolabeled sucrose covalently linked to protein: a device for quantifying degradation of plasma proteins catabolized by lysosomal mechanisms. J Biol Chem. 1979;254:6876–6879.[Abstract/Free Full Text]
  20. Robenek H, Schmitz G, Assmann G. Topography and dynamics of receptors for acetylated and malondialdehyde-modified low density lipoproteins in the plasma membrane of mouse peritoneal macrophages as visualized by colloidal gold in conjunction with surface replicas. J Histochem Cytochem. 1984;32:1017–1027.[Abstract]
  21. Basu SK, Goldstein JL, Anderson GW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;73:3178–3182.[Abstract/Free Full Text]
  22. Bergendorff A, Uvnäs B. Storage of 5-hydroxytryptamine in rat mast cells: evidence for an ionic binding to carboxyl groups in a granule heparin-protein complex. Acta Physiol Scand. 1972;84:320–331.[Medline] [Order article via Infotrieve]
  23. Brown MS, Ho YK, Goldstein JL. The cholesteryl ester cycle in macrophage foam cells: continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters. J Biol Chem. 1980;255:9344–9352.[Free Full Text]
  24. Kritharides L, Jessup W, Gifford J, Dean RT. A method for defining the stages of low-density lipoprotein oxidation by the separation of cholesterol- and cholesteryl ester-oxidation products using HPLC. Anal Biochem. 1993;213:79–89.[Medline] [Order article via Infotrieve]
  25. Sorger T, Germinario RJ. A direct solubilization procedure for the determination of DNA and protein in cultured fibroblast monolayer. Anal Biochem. 1983;131:254–256.[Medline] [Order article via Infotrieve]
  26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]
  27. Bartold PM, Page RC. A microdetermination method for assaying glycosaminoglycans and proteoglycans. Anal Biochem. 1985;150:320–324.[Medline] [Order article via Infotrieve]
  28. Miyazawa K, Fukuyama J, Misawa K, Hamano S, Ujiie A. Tranilast antagonizes angiotensin II and inhibits its biological effects in vascular smooth muscle cells. Atherosclerosis. 1996;121:167–173.[Medline] [Order article via Infotrieve]
  29. Soter NA, Austen KF, Wasserman SI. Oral disodium cromoglycate in the treatment of systemic mastocytosis. N Engl J Med. 1979;301:465–469.[Abstract]
  30. Barnes PJ. Asthma as an axon reflex. Lancet. 1986;1:242–244.[Medline] [Order article via Infotrieve]
  31. Hägermark Ö, Hökfelt T, Pernow B. Flare and itch induced by substance P in human skin. J Invest Dermatol. 1978;71:233–235.[Medline] [Order article via Infotrieve]
  32. Kaplan AP, Beaven MA. In vivo studies of the pathogenesis of cold urticaria, cholinergic urticaria, and vibration-induced swelling. J Invest Dermatol. 1976;67:327–332.[Medline] [Order article via Infotrieve]
  33. Rus HG, Niculescu F, Constantinescu E, Cristea A, Vlaicu R. Immunoelectron-microscopic localization of the terminal C5b-9 complement complex in human atherosclerotic fibrous plaque. Atherosclerosis. 1986;61:35–42.[Medline] [Order article via Infotrieve]
  34. Füreder W, Agis H, Willheim M, Bankl HC, Maier U, Kishi K, Müller MR, Czerwenka K, Radaszkiewicz T, Butterfield JH, Klappacher GW, Sperr WR, Oppermann M, Lechner K, Valent P. Differential expression of complement receptors on human basophils and cast cells: evidence for mast cell heterogeneity and CD88/C5aR expression on skin mast cells. J Immunol. 1995;155:3152–3160.[Abstract]
  35. Sedgwick JD, Holt PG, Turner KJ. Production of a histamine-releasing lymphokine by antigen- or mitogen-stimulated human peripheral T cells. Clin Exp Immunol. 1981;45:409–418.[Medline] [Order article via Infotrieve]
  36. Thueson DO, Speck LS, Lett-Brown MA, Grant JA. Histamine-releasing activity (HRA), I: production by mitogen- or antigen-stimulated human mononuclear cells. J Immunol. 1979;123:626–632.[Abstract/Free Full Text]
  37. Johnson JL, Jackson CL, Angelini GD, George SJ. Activation of matrix-degrading metalloproteinases by mast cell proteases in atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 1998;18:1707–1715.[Abstract/Free Full Text]
  38. Church MK, Lowman MA, Rees PH, Benyon RC. Mast cells, neuropeptides and inflammation. Agents Actions. 1989;27:8–16.[Medline] [Order article via Infotrieve]
  39. Hachisuka H, Nomura H, Sakamoto F, Mori O, Okubo K, Sasai Y. Effect of antianaphylactic agents on substance-P induced histamine release from rat peritoneal mast cells. Arch Dermatol Res. 1988;280:158–162.[Medline] [Order article via Infotrieve]
  40. Reichl D, Postiglione A, Myant NB, Pflug JJ, Milis GL. The lipids and lipoproteins of human peripheral lymph, with observations on the transport of cholesterol from plasma and tissues into lymph. Clin Sci Mol Med. 1973;49:419–426.
  41. Smith EB. Transport, interactions and retention of plasma proteins in the intima: the barrier function of the internal elastic lamina. Eur Heart J. 1990;11(suppl E):72–81.
  42. Kokkonen JO, Lindstedt KA, Kovanen PT. Role of mast cell proteases and proteoglycans in lipoprotein metabolism. In: Gaughey GH, ed. Mast Cell Proteases in Immunology and Biology. New York, NY: Marcel Dekker; 1995:257–287.
  43. Kovanen PT, Kokkonen JO. Modification of low density lipoproteins by secretory granules of rat serosal mast cells. J Biol Chem. 1991;266:4430–4436.[Abstract/Free Full Text]
  44. Kokkonen JO, Kovanen PT. Proteolytic enzymes of mast cell granules degrade low density lipoproteins and promote their granule-mediated uptake by macrophages in vitro. J Biol Chem. 1989;264:10749–10755.[Abstract/Free Full Text]
  45. Galli SJ. New concepts about the mast cell. N Engl J Med. 1993;328:257–265.[Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ma, H.
Right arrow Articles by Kovanen, P. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ma, H.
Right arrow Articles by Kovanen, P. T.
Related Collections
Right arrow Pathophysiology
Right arrow Lipid and lipoprotein metabolism

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2000 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.