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. 1995;15:811-819

This Article
Right arrow Abstract Freely available
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 HighWire
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.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:811-819.)
© 1995 American Heart Association, Inc.

Articles

IgE-Dependent Generation of Foam Cells: An Immune Mechanism Involving Degranulation of Sensitized Mast Cells With Resultant Uptake of LDL by Macrophages

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Because a role has been suggested for IgE in cardiovascular diseases and for mast cells in cholesterol accumulation within the macrophages of atherosclerotic lesions, we examined mast cell–macrophage interactions in vitro by using rats with high serum levels of IgE antibodies. The rats were immunized with an antigen (ovalbumin) and adjuvant (Bordetella pertussis vaccine) to provoke synthesis of IgE and to sensitize their mast cells, ie, to allow the IgE to bind to the high-affinity IgE receptors on the mast cell surfaces. On addition of the ovalbumin to suspensions of mast cells isolated from the peritoneal cavity of the immunized rats, the mast cells responded by exocytosing their heparin-proteoglycan–containing granules. When IgE-bearing peritoneal mast cells were cocultured with peritoneal macrophages (also from the immunized rats) in a medium enriched in LDL, addition of ovalbumin to the incubation medium triggered a dose-dependent release of granules and a dose-dependent increase in the rate of LDL uptake by the macrophages. In contrast, ovalbumin had no effect on LDL uptake if the cultures contained only macrophages or if the mast cells and macrophages were from nonimmunized rats. Thus, the sequence of events leading to enhanced uptake of LDL by macrophages depended wholly on IgE-dependent degranulation of the sensitized mast cells. With the aid of gold-labeled LDL we demonstrated that the exocytosed mast cell granules had bound LDL particles and carried them into the macrophages, with subsequent formation of foam cells. The current series of experiments delineates a novel immunologic mechanism for the formation of macrophage foam cells and assigns a potentially atherogenic role to mast cell–bound IgE antibodies.


Key Words: cardiovascular diseases • foam cells • IgE antibodies • LDL • mast cells


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The development of cardiovascular disease is associated with elevated levels of IgE, suggesting that IgE-mediated events play a role in the pathogenesis of these diseases.1 2 3 4 Atherosclerosis is the most prevalent and prominent process in the pathogenesis of cardiovascular diseases, and it affects the innermost layer of the arterial wall, the intima.5 In fatty streaks, which are the earliest gross atherosclerotic lesions, LDL-derived cholesterol accumulates in intimal macrophages, producing cholesterol-filled foam cells.6 7 8 Macrophage foam cells are also present in the late atherosclerotic lesions, the atheromas, mostly in the growing margins or the shoulder regions.9

Both the normal and atherosclerotic intima contain, in addition to macrophages, blood-borne immunologically active cells of another type, mast cells.10 11 12 13 14 Mast cells often reside in close vicinity with macrophage foam cells.12 13 In the fatty streaks of human aortic15 and coronary16 intimas the numbers of mast cells are significantly greater than in the normal intima, findings that are compatible with the notion that mast cells may participate in the formation of macrophage foam cells during atherogenesis. Findings from this laboratory have, in fact, assigned a role for stimulated rat mast cells in the formation of macrophage foam cells.17 18 19 20 The studies reveal that in vitro in the presence of LDL, stimulation of rat serosal mast cells with compound 48/80 (a noncytotoxic substance of low-molecular weight capable of specifically stimulating mast cells) leads to binding of LDL to exocytosed cytoplasmic granules, ie, the granule remnants, and to proteolytic modification of the bound LDL by the granule-remnant neutral proteases chymase and carboxypeptidase A. Such granule remnant–bound proteolytically modified LDL particles are then taken up by cocultured macrophages, as the granule remnants are phagocytosed by the macrophages. We have also noted that the neutral proteases of exocytosed granules proteolyze HDL3, thereby lessening their ability to induce efflux of cholesterol from the cholesterol-loaded macrophages.21 Taken together, these observations suggest that exocytosed mast cell proteoglycans and neutral proteases play a role in the formation of macrophage foam cells.

The classic example of mast cell stimulation is their activation by IgE.22 In IgE-mediated degranulation, the relevant antigen (allergen) is bound by two or more of the IgE molecules bound to receptors with high affinity for IgE (Fc{varepsilon}R1) on the mast cell surface ("sensitized mast cells"). It is this cross-linkage of cell-bound IgE with bridging of IgE receptors that triggers mast cell degranulation. To study possible mechanisms for IgE-dependent formation of macrophage foam cells, we used an experimental model in which rats are sensitized by immunizing them to ovalbumin with Bordetella pertussis vaccine as adjuvant. Rats so immunized are known to produce high levels of the IgE antibody against the antigen (ovalbumin).23 Here we show that antigenic stimulation of sensitized mast cells leads to increased uptake of LDL by cocultured macrophages and thus observe an IgE-dependent mechanism involved in the genesis of foam cells.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animals and Reagents
Male Wistar rats (age, 9 to 20 weeks; weight, 200 to 350 g) were used throughout the study. The rats were obtained from the Laboratory Animal Center of the University of Helsinki. Ovalbumin, heparin, and bovine serum albumin (BSA) were from Sigma; aluminum hydroxide gel and lactic acid were from Merck; mouse monoclonal anti-rat heavy chain IgE was from Serotec; RPMI-1640 culture medium supplemented with 25 mmol/L HEPES and Dulbecco's phosphate-buffered saline (PBS) were from GIBCO; Eagle's basal medium with Earle's salts and 1.68 g/L sodium bicarbonate without glutamine (EBME) was from Flow Laboratories; and [U-14C]sucrose (200 Ci/mol) was from Amersham International. Pertussis vaccine and fibronectin were kind gifts from the National Public Health Institute of Finland and the Finnish Red Cross Blood Transfusion Service, Helsinki, respectively. Plastic dishes were from Falcon.

Immunization of Rats and Active Sensitization of Mast Cells
Rats, when they weighed about 200 to 250 g, were given a single injection of ovalbumin 1 mg IM as antigen in 1 mL physiological saline containing 200 mg aluminum hydroxide gel. To enhance the production of IgE antibodies, inactivated Bordetella pertussis organisms (2x1010 in 0.5 mL physiological saline) were injected subcutaneously as adjuvant at the same time as the antigen.24 At various times after the start of immunization, rats were killed to obtain immune serum and sensitized peritoneal mast cells. The immune serum was either used immediately or stored at -70°C and later thawed once and used for the experiments. Mast cells were used for the experiments immediately after their isolation.

Isolation of Peritoneal Macrophages and Mast Cells
Peritoneal macrophages and mast cells were isolated from sensitized or nonsensitized rats essentially as described for isolation of peritoneal cells from normal rats.25 Briefly, after injection into the peritoneal cavity of 20 mL PBS containing 0.5 mg/mL BSA, 0.05 mg/mL heparin, and 5.6 mmol/L glucose, pH 7.3, the cells on the surfaces of the peritoneal cavity were collected by lavage. The cells were washed by sedimenting and resuspended in culture medium A (RPMI-1640 supplemented with 10 mg/mL BSA, 5% fresh autologous rat serum, 100 IU/mL penicillin, and 2 mmol/L L-glutamine). To allow the macrophages to adhere to plastic, the cells were seeded into plastic microtiter wells (24-well plates) or into plastic Petri dishes (150x15 mm) and incubated in a humidified incubator (5% CO2 in air) at 37°C for 2 hours. After incubation, nonadherent cells were removed, washed once by centrifugation in PBS, and resuspended in culture medium. Analysis of the cell suspension by alcian blue staining revealed that it contained mast cells of 90% to 95% purity.

Preparation of Immune Serum and Passive Sensitization of Peritoneal Mast Cells
For passive sensitization of mast cells, immune serum was obtained from rats 3 to 4 weeks after immunization. The ability of immune serum to sensitize mast cells is highest during this interval; mast cells exposed for several hours to such sera (ie, passive sensitization), when challenged with 4 µg/mL ovalbumin, released at least 40% of their histamine. To passively sensitize mast cells, peritoneal cells were obtained from nonimmunized rats as described above. The cells (mast cells and macrophages) were resuspended in PBS containing 50% (vol/vol) immune (anti-ovalbumin) serum and incubated at 37°C for 3 hours. After incubation, the cells were washed twice with PBS by centrifugation, resuspended in culture medium, transferred to culture dishes (to which the macrophages but not the mast cells became attached), and used for the experiments.

Dissociation of IgE From Actively Sensitized Mast Cells
Surface-bound IgE was dissociated from mast cells obtained from immunized rats by the lactic acid elution method of Pruzansky et al.26 In each assay {approx}10x105 actively sensitized mast cells were incubated at 15°C for 10 minutes in 100 µL of 10 mmol/L lactic acid solution, pH 3.9. After incubation, 1 mL PBS containing 10 mg/mL BSA was added, and the mast cells (stripped of IgE by lactic acid) were washed with 1 mL PBS, sedimented by centrifugation at 400g for 5 minutes at 4°C, resuspended in 200 µL PBS, and used for experiments.

Histamine Release From Mast Cells
Actively or passively sensitized mast cells (5x104) were preincubated at 37°C for 5 minutes in 200 µL PBS, after which the indicated amounts of stimulants (ovalbumin or monoclonal anti-rat IgE) were 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 cells were sedimented by centrifugation at 400g for 5 minutes at 4°C. The histamine contents of both supernatant and mast cells were determined fluorometrically according to Bergendorff and Uvnäs27 with modifications.28 Histamine release was expressed as a percentage of the total histamine content of the mast cells.

Isolation of LDL and Preparation of [14C]Sucrose-LDL and Colloidal Gold–LDL Conjugates
Human LDL (d=1.019 to 1.050 g/mL) was isolated from plasma by sequential ultracentrifugation in the presence of 3 mmol/L Na2-EDTA.28 The isolated LDL was labeled with [14C]sucrose to yield specific activities in the range of 20 to 30 dpm/ng LDL protein.29 Colloidal gold particles (15 nm) were prepared and conjugated with LDL as described by Robenek et al.30 Gold-LDL preparations were examined by transmission electron microscopy, using negative staining, and were found to contain 3 to 5 LDL molecules per gold particle. The concentration of the LDL preparation is expressed in terms of its protein concentration.

Uptake of [14C]Sucrose-LDL by Cultured Macrophages
Macrophage monolayers (1x106 cells/well) prepared from immunized or nonimmunized rats received actively or passively sensitized mast cells (1x105) in 1 mL medium B (EBME containing 10 mg/mL BSA and 100 IU/mL penicillin). The cells were incubated (in a 5% CO2 atmosphere) for 1 hour at 37°C, after which the indicated amounts of ovalbumin were added, and incubation was continued for 30 minutes. Finally, 15 µg [14C]sucrose-LDL was added, and incubation was continued for the indicated periods. At the end of incubation the medium was removed, and the macrophage monolayers were washed twice with 1 mL PBS containing 10 mg/mL BSA and 10 mg/mL heparin and then twice with 1 mL PBS. Histamine was determined from a 200-µL aliquot of the medium from which the cells had been removed by centrifugation. The macrophage monolayers were dissolved in 0.2N NaOH, and their 14C radioactivity and protein contents were determined.

Uptake of Gold-LDL by Macrophages
After incubation with stimulated, actively sensitized mast cells in the presence of gold-LDL, 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. After a 2-day polymerization, the Epon blocks were sectioned horizontally along the cell layer. 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 at the Department of Electron Microscopy, University of Helsinki, Finland.

Oil Red O Staining of Lipid Droplets in Macrophages
Mixtures of mast cells and macrophages from sensitized rats were incubated in microtiter wells in which glass coverslips were placed. The mast cells were stimulated with antigen (ovalbumin) in the presence of unlabeled LDL and incubated as described for the experiments in which uptake of [14C]sucrose-LDL by macrophages was measured. After incubation, the macrophage monolayers were washed, fixed with 4% formaldehyde, stained with oil red O, and counterstained with Harris hematoxylin.

Other Assays
Protein was determined by the procedure of Lowry et al31 with BSA as standard. 14C radioactivity was measured in a liquid scintillation counter (1215 Rackbeta) and expressed in disintegrations per minute.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Peritoneal mast cells were obtained from ovalbumin-immunized rats (actively sensitized mast cells) and incubated in the presence of increasing concentrations of ovalbumin. A dose-dependent release of histamine ensued, ie, there was a degranulation of mast cells (Fig 1ADown). If mast cells from nonimmunized rats were passively sensitized by incubating them in serum from immunized rats and then challenged with ovalbumin, a similar dose response of histamine release was observed (Fig 1BDown). Maximum histamine release was observed at an ovalbumin concentration of 2 µg/mL. As control ("nonsensitized") cells for the actively and passively sensitized mast cells, we used mast cells obtained from nonimmunized rats and left either untreated (Fig 1ADown and 1CDown) or incubated with nonimmune serum (Fig 1BDown and 1DDown). In neither case did addition of ovalbumin cause any significant histamine release above the level of spontaneous release ({approx}5%) observed in the absence of antigen. The response to ovalbumin was rapid; histamine release reached the maximum level within 1 minute in both the actively and passively sensitized mast cells (Fig 1CDown and 1DDown). In light of the above results, in subsequent experiments both actively and passively sensitized mast cells were incubated in the presence of 4 µg/mL ovalbumin for 10 minutes to stimulate mast cells maximally and allow complete degranulation.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Line graphs showing antigen-induced degranulation of actively and passively sensitized rat peritoneal mast cells as a function of antigen concentration (A and B) and time (C and D). Mast cells (5x104) were incubated at 37°C for 5 minutes in 200 µL phosphate-buffered saline, after which ovalbumin was added to give the indicated concentrations (A and B) or 8 µg/mL (C and D). The incubations were continued for 10 minutes (A and B) or for the indicated times (C and D). After incubation, the cells were sedimented by centrifugation, and the contents of histamine in the supernatants and sedimented cells were determined. Histamine release is expressed as a percentage of the total histamine present in the cells prior to stimulation.

To follow the appearance and persistence of mast cell sensitization in vivo (active sensitization), peritoneal mast cells were obtained at various times after immunization. The mast cells were challenged with ovalbumin as described above, and histamine release was measured. To follow the appearance and persistence of mast cell–sensitizing antibodies (IgE) in serum, serum was obtained at various times after immunization, and mast cells from normal rats were passively sensitized with these samples of sera. Finally, the passively sensitized mast cells were challenged with ovalbumin, and histamine release was measured. The results of these experiments (Fig 2Down) showed that mast cells became sensitized (ie, mast cell–sensitizing antibodies appeared in the serum) as little as 2 weeks after antigen injection and that with both actively and passively sensitized mast cells the peak responses were reached after {approx}1 month. After that, the ability of the immune serum to sensitize mast cells rapidly declined, whereas the ability of the actively sensitized mast cells to respond to the relevant antigen remained near the peak level throughout the 2-month observation period (4 to 12 weeks).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Line graph showing time courses of sensitization of peritoneal mast cells and of the generation of anti-ovalbumin antiserum in rats immunized with ovalbumin. Rats were immunized by subcutaneous injection of ovalbumin as antigen and Bordetella pertussis vaccine as adjuvant. Rats were killed at the indicated times after injection, and peritoneal mast cells and immune sera were prepared as described in "Methods." Actively sensitized mast cells ({bullet}——{bullet}) were incubated in the presence of 4 µg/mL ovalbumin, and histamine release was determined as described in the legend to Fig 1Up. Passively sensitized mast cells ({circ}—{circ}) (mast cells and macrophages) obtained from nonsensitized rats by peritoneal lavage were incubated in the presence of immune serum, the mast cells were separated and incubated with 4 µg/mL ovalbumin, and histamine release was determined as described above.

The above responses were as expected for the IgE-dependent response in this rat model, in which antibodies of this class disappear faster from serum than from tissues, in which they become fixed when bound to the high-affinity receptors for IgE on mast cell surfaces.23 To obtain more direct evidence for the presence of IgE in serum and on the actively sensitized mast cells, we conducted the following experiments (Fig 3Down). Like human IgE, rat IgE is destroyed by heating at 56°C.23 Such treatment of the immune serum totally abolished its ability to sensitize mast cells (Fig 3ADown). Moreover, treatment of actively sensitized mast cells with lactic acid, a compound that releases IgE from its high-affinity receptors,26 rendered the mast cells nonresponsive to the relevant antigen (ovalbumin) (Fig 3BDown). In a control experiment, immune serum, when added to the cells after lactic acid treatment, partially restored their responsiveness to antigen challenge, demonstrating that the cells were still viable after treatment with lactic acid (not shown). Finally, addition of monoclonal antibody against rat IgE triggered histamine release from the actively sensitized mast cells, demonstrating that these cells did bear IgE on their surfaces (Fig 3CDown). We concluded that in this experimental system degranulation of both passively and actively sensitized mast cells with the relevant antigen were events mediated by IgE.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Line graphs showing (A) effect of heat inactivation of immune serum on its ability to passively sensitize mast cells, (B) effect of lactic acid treatment of actively sensitized mast cells on the ability of specific antigen to induce their degranulation, and (C) degranulation of actively sensitized mast cells by anti-rat IgE. A, Cells from peritoneal lavage of nonsensitized rats were incubated in the presence of immune serum ({bullet}——{bullet}) obtained from rats 4 weeks after injection of antigen, the mast cells were isolated, and 5x104 mast cells were incubated for 10 minutes in the presence of the indicated concentrations of ovalbumin. In parallel experiments ({circ}—{circ}), the immune serum was heated at 56°C for 30 minutes before use. B, Actively sensitized mast cells (5x104) before ({bullet}——{bullet}) and after ({circ}—{circ}) lactic acid treatment were incubated in the presence of the indicated concentrations of ovalbumin for 10 minutes. C, Actively sensitized or nonsensitized mast cells (5x104) were incubated in the presence of the indicated concentrations of monoclonal anti-rat IgE for 30 minutes. In each experiment, the reaction was stopped, and histamine was determined as described in "Methods."

To investigate the ability of the IgE-mediated stimulation of mast cells to induce uptake of LDL by macrophages, mast cells and macrophages obtained from the peritoneal cavity of immunized rats were cocultured in the presence of LDL. When increasing amounts of ovalbumin were added to the culture medium, increasing amounts of histamine were released by the mast cells (Fig 4ADown). Measurement of LDL uptake by the cocultured macrophages revealed that the rate of uptake closely followed the increase in histamine release by the mast cells (Fig 4BDown). Similar results were obtained when mast cells were passively sensitized with immune serum (Fig 4CDown and 4DDown). The similarity of the rates of LDL uptake by macrophages from immunized rats (active sensitization of mast cells) and from nonimmunized rats (passive sensitization of mast cells) showed that even if the immunization program did cause long-term activation of macrophages, this was not essential for the increased rate of uptake of LDL. The above observation concerning the passive sensitization system also excluded the possibility that the macrophages had been activated by ovalbumin-sensitized T lymphocytes (possibly contaminating the preparation of actively sensitized mast cells), which might have released cytokines upon addition of ovalbumin. If the mast cells in the incubation system were nonsensitized, neither antigen-induced histamine release by mast cells nor antigen-induced uptake of LDL by macrophages was observed. Since rat macrophages possess low-affinity receptors for IgE (Fc{varepsilon}R2) that may activate these cells by IgE-dependent mechanisms,32 we conducted a control experiment in which we examined the effect of ovalbumin (0 to 8 µg/mL) on LDL uptake by peritoneal macrophages from immunized rats (5 and 15 weeks after immunization). Addition of ovalbumin had no effect on the rate of LDL uptake by the macrophages, confirming that in this in vitro system the IgE-dependent acceleration of LDL uptake by macrophages took place only in the presence of sensitized mast cells.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 4. Line graphs showing antigen-induced degranulation of actively (A) and passively (C) sensitized mast cells and uptake of [14C]sucrose-LDL by macrophages cocultured with actively (B) and passively (D) sensitized mast cells. Each dish contained a monolayer of rat peritoneal macrophages (1x106 cells/well) and mast cells (1x105) in 1 mL medium B. In each experiment the macrophages and mast cells were derived from the same immunized or nonimmunized rats. Actively sensitized mast cells (and macrophages) were obtained from rats 5 weeks after immunization, and passively sensitized mast cells (and macrophages) were treated with immune serum obtained from rats 4 weeks after immunization. After incubation at 37°C for 1 hour, ovalbumin was added to give the indicated concentrations, and the incubations were continued for 30 minutes. Finally, 15 µg [14C]sucrose-LDL (21 dpm/ng in B and 30 dpm/ng in D) was added, and incubation was continued for 14 hours. Histamine release from mast cells and uptake of LDL by macrophages were determined as described in "Methods." A value of histamine release from mast cells and a value uptake of LDL by macrophages in the absence of ovalbumin (blank values) have been subtracted from the respective values obtained in the presence of varying concentrations of ovalbumin.

The quantitative relation between the extent of mast cell degranulation and the increase in LDL uptake was consistent with a model in which exocytosed mast cell granules (ie, granule remnants) mediate uptake of LDL by cocultured macrophages.19 Involvement of granule remnants was corroborated by inclusion in the incubation medium of fibronectin, a molecule capable of competing with LDL for binding to granule remnants.33 It was found that fibronectin strongly inhibited the mast cell–induced uptake of LDL by macrophages, evidently by inhibiting the binding of LDL to the granule remnants (Fig 5Down).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 5. Line graph showing time course of LDL uptake by macrophages cocultured with actively sensitized antigen-stimulated mast cells. Each well received a mixture of peritoneal cells in 1 mL medium B (2x105 mast cells and 2.5x106 macrophages obtained by peritoneal lavage of five immunized rats 5 weeks after immunization). After incubation at 37°C for 1 hour, mast cells were stimulated by addition of 8 µg/mL ovalbumin, and incubation was continued for 10 minutes to allow completion of mast cell degranulation. Fibronectin was added to a series of dishes (final concentration, 200 µg/mL; {circ}—{circ}). Five minutes after addition of fibronectin, each dish received 15 µg [14C]sucrose-LDL (23 dpm/ng). After incubation at 37°C for the indicated times, the medium was removed, and uptake of LDL by macrophages was determined as described in "Methods."

To obtain direct evidence for the ability of the granule remnants to bind LDL and carry it into macrophages, the LDL-bearing granule remnants were visualized with gold-labeled LDL. LDL particles bound to colloidal gold effectively bind to mast cell granule remnants, as do unlabeled LDL particles.18 33 Gold-labeled LDL was added to the incubation medium, and the mast cells were stimulated with ovalbumin. Four hours after stimulation the cells were fixed and prepared for transmission electron microscopy. Fig 6ADown shows three extracellularly located granule remnants coated with gold-labeled LDL. A macrophage is in the process of phagocytosing one of these remnants. In Fig 6BDown, phagocytosis of the granule remnants is more advanced. Thus, in the cytoplasm of the macrophages, two phagosomes can be seen, one containing one and the other containing two LDL-bearing granule remnants. In addition, the extracellular space is seen to contain gold-LDL aggregates, some isolated, others clearly connected with the granule remnants, and still others being phagocytosed (Fig 6ADown). Some of the phagosomes contain gold-LDL aggregates without granule remnants (Fig 6BDown). These gold-LDL aggregates consist of insoluble complexes formed between LDL and the soluble heparin proteoglycans released from the exocytosed mast cell granules.34 These complexes, in contrast to the LDL-loaded granule remnants, are ingested by macrophages via scavenger receptor–mediated phagocytosis.20 34



View larger version (128K):
[in this window]
[in a new window]
  		Figure 6. Photomicrographs showing uptake of gold-LDL by macrophages (MØ). Macrophages (6x106) and mast cells (3x105) obtained from rats 4 weeks after immunization were cocultured at 37°C for 1 hour in 1.5 mL medium B. The mast cells were then stimulated with ovalbumin (8 µg/mL), and incubation was continued for 10 minutes to allow completion of mast cell degranulation. Finally, gold-LDL (30 µg/mL) was added, and incubation continued for 4 hours. After incubation the medium was removed, and the macrophage monolayers were washed twice with phosphate-buffered saline and fixed with 2% glutaraldehyde. The blocks were sectioned at 60 nm, and the ultramicrotome sections were viewed and photographed by using an electron microscope (original magnification x10 000). Gr indicates granule remnant; bar=500 nm.

The phagocytosed granule remnant–bound LDL particles become rapidly degraded by the macrophages while the rate of cholesteryl ester synthesis in these cells simultaneously increases.17 This pathway parallels that described in macrophages incubated with acetylated LDL,35 which suggests that cytoplasmic cholesteryl esters should accumulate in this model also. In a search for the presence of foam cells, macrophages were stained with oil red O after coculture with antigen-stimulated mast cells for 24 hours in the presence of LDL. Such macrophages contained numerous oil red O–positive cytoplasmic droplets (Fig 7ADown). Macrophages cocultured with unstimulated mast cells also contained some lipid droplets (Fig 7CDown), apparently due to spontaneous degranulation occurring during prolonged incubation of mast cells. However, counts of the lipid droplets revealed that macrophages incubated with stimulated mast cells contained significantly more lipid droplets than those cultured with unstimulated mast cells (Fig 7BDown and 7DDown).



View larger version (2K):
[in this window]
[in a new window]
  		Figure 7. A and C, Photomicrographs showing oil red O staining of macrophage foam cells. Macrophages (1.5x106) and mast cells (2x105) obtained from rats 6 weeks after immunization were cocultured at 37°C for 1 hour in 1 mL medium B. The mast cells were either (A) stimulated with ovalbumin (20 µg/mL) or (C) left unstimulated. After 10 minutes, LDL (200 µg/mL) was added to all dishes, and incubation was continued for 24 hours. After incubation the macrophage monolayers were washed, fixed with 4% formaldehyde, and stained with oil red O as described in "Methods" (original magnification x400). Below each photomicrograph is the corresponding histogram (B and D) of the number of oil red O–positive droplets per cell.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present experiments demonstrate that IgE-mediated stimulation of rat peritoneal mast cells leads to enhanced uptake of LDL by macrophages and eventually to formation of foam cells. This newly described immunologic mechanism of foam cell formation depends on the ability of exocytosed mast cell granules (ie, granule remnants) to carry LDL into the macrophages. This carrier mechanism is efficient: a single stimulation of mast cells is sufficient for the generation of macrophage foam cells in vitro. The efficiency must depend, at least in part, on the high content of granules in mast cells and the high capacity of the exocytosed granules to bind LDL. Thus, each rat serosal mast cell contains, on average, 1000 secretory granules, and each granule can bind up to 5000 to 10 000 LDL particles.18 The mechanism of LDL uptake is similar to that for mast cells cocultured with macrophages in LDL-enriched medium and stimulated nonimmunologically with compound 48/80.17 The similarity between the immunologically and nonimmunologically triggered mast cell–dependent mechanisms identifies "the granule remnant carrier system"19 as the common end pathway in which the mast cell–macrophage interaction may ultimately lead to formation of macrophage foam cells. The similarity suggests that any stimulus causing mast cells to degranulate would be sufficient to initiate the sequence of events described here. Indeed, the atherosclerotic human arterial intima appears to contain a wide variety of physiological agents capable of triggering mast cells to extrude their granules. These include activated complement36 and histamine-releasing factors that are secreted by T lymphocytes37 and macrophages,38 two cell types also present in the atherosclerotic intima.39 40 Interestingly, some of the locally produced histamine-releasing factors also require the presence of IgE on the mast cell surface to cause histamine release.41 42 43 The best evidence so far for actual stimulation of mast cells in the human aortic intima and coronary intima comes from light and electron microscopic studies of autopsy material in which partially degranulated mast cells and extracellularly located granule remnants have been observed.15 16 Indeed, in these human coronary atheromas 90% of all mast cells were found to be stimulated to exocytose some of their cytoplasmic secretory granules. Thus, the mast cells of the human atherosclerotic arterial intima reside in an immunologically active area, where they are exposed to many potential local stimulants and become activated by these stimuli, some of which appear to be IgE-dependent.

As discussed above, the areas of the arterial intima where foam cell formation occurs appear to provide conditions suitable for efficient exocytosis of mast cells. These same atherosclerosis-prone areas also appear to provide the conditions necessary for efficient phagocytosis of the granules released, for they contain large numbers of macrophages44 and smooth muscle cells of the synthetic phenotype,45 the former being specialized as phagocytes and the latter also having a high phagocytotic capacity.46 47 It would be valuable to know whether the phagocytotic uptake of LDL-loaded granule remnants is regulated by the various cytokines and other soluble mediators secreted by stimulated mast cells.48 However, both isolated granule remnants (which have lost their soluble mediators) and stimulated mast cells (secreting both granules and soluble mediators) are able to produce macrophage foam cells if LDL is present in the incubation medium.17 These observations demonstrate that, at least in vitro, the soluble factors secreted by stimulated mast cells are neither necessary for nor prevent mast cell–dependent foam cell formation.

For an IgE-mediated mechanism of foam cell formation to operate in the human arterial intima, the IgE molecules must reach the intimal fluid and be bound to the intimal mast cells. In addition, the specific antigens (allergens) against which the IgE molecules are directed must reach the intimal fluid and bind to IgE on the sensitized intimal mast cells. What conditions are required for encounters between IgE antibody molecules and the specific antigen on the surface of an intimal mast cell? IgE and its receptors (originally evolved against parasites) are directed against exogenous antigens that invade the organism through the skin or mucosal surfaces of the body.49 Accordingly, IgE-secreting B cells are abundant in the skin, lungs, and gut,50 and it is in these barrier tissues that mast cells reach their highest densities.48 Indeed, local production of antigen-specific IgE has been demonstrated in the respiratory mucosa without evidence of this antibody in the serum.51 However, in allergic individuals high levels of circulating IgE are usually present, reflecting overproduction of IgE in response to common environmental antigens.52 In these individuals, the circulating IgE molecules can reach and sensitize the mast cells in various tissues. External antigens can also reach the circulation and various tissues. Thus, experimental and clinical studies have shown that a fraction of the ingested food antigens (undigested proteins) can be absorbed from the intestine53 54 55 and may trigger an allergic response in another barrier tissue, such as the skin, or the mucosal surface of the lungs, eyes, or nose.55 56 Accordingly, in allergic individuals specific antigens may enter one barrier tissue and be targeted to another barrier tissue. Moreover, an "allergy of the abdominal organs" (including the peritoneum, a site heavily populated with mast cells) has been described in the rhesus monkey, suggesting that allergens are disseminated to other targets besides the barrier tissues.57 There remains the challenging task of demonstrating that in allergic individuals one such target tissue is the arterial intima.

The hypothesis linking IgE-mediated stimulation of mast cells in the human coronary intima to coronary artery disease has so far received only limited support. The few clinical or epidemiological observations available that link elevated serum IgE levels and coronary artery disease1 2 3 4 deal with the late symptomatic phase of the disease, when the fatty streak lesions have evolved into complicated atheromas. Thus, the patients in the studies cited were suffering from unstable angina or myocardial infarction. Mast cells, which transform macrophages into foam cells, may contribute to these acute events of coronary atherosclerosis by causing plaque destabilization. Thus, in human atherosclerotic lesions the number of degranulated mast cells is especially increased in specific areas of atheromas, the shoulder regions, that are susceptible to atheromatous rupture.15 16 These regions contain large numbers of macrophage foam cells that have been induced to express matrix-degrading enzymes such as stromelysin.58

Taken together, the current findings suggest a novel immune mechanism of atherogenesis, the critical event being IgE-dependent mast cell stimulation. Immunologic arterial injury has long been considered to play an important role in the pathogenesis of atherosclerosis, especially in clinical settings such as the coronary arteritis seen in lupus erythematosus.59 60 61 The concept of immunologic arterial injury has obtained support from experimental work conducted in rabbits that demonstrates that the arterial endothelium may be damaged by circulating immune complexes formed between antigens and IgG antibodies.62 However, of the serum immunoglobulins, IgE very rarely participates in circulating immune complexes. Thus, the IgE-mediated mechanisms are likely to be different and to be restricted to the cellular events triggered by interaction between receptor-bound IgE molecules and their specific antigens. Very recently, a central role in the pathogenesis of atherosclerosis has been assigned to immune mechanisms, the critical functions being now attributed to macrophages, T lymphocytes, smooth muscle cells, endothelial cells, and platelets.63 The present demonstration of immunologically triggered mast cell–dependent foam cell formation adds a new cell type, the mast cell, and a new class of immunoglobulins, IgE, to the complex immunologic scenario of atherogenesis.

Received September 5, 1994; accepted March 22, 1995.


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

  1. Criqui MH, Lee ER, Hamburger RN, Klauber MR, Coughlin SS. IgE and cardiovascular disease: results from a population-based study. Am J Med. 1987;82:964-968. [Medline] [Order article via Infotrieve]
  2. Szczeklik A, Sladek K, Szczerba A, Dropinski J. Serum immunoglobulin E response to myocardial infarction. Circulation. 1988;77:1245-1249. [Abstract/Free Full Text]
  3. Criqui MH. Serum immunoglobulin E after to myocardial infarction. Circulation. 1988;78:1322. Letter. [Free Full Text]
  4. Korkmaz ME, Oto A, Saraclar Y, Oram E, Oram A, Ugurlu S, Karamehmetoglu A, Karaagaoglu E. Levels of IgE in the serum of patients with coronary arterial disease. Int J Cardiol. 1991;31:199-204. [Medline] [Order article via Infotrieve]
  5. Stary HC, Blankenhorn DH, Chandler AB, Glagov S, Insull W Jr, Richardson M, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition of the intima of human arteries and of its atherosclerosis-prone regions. Circulation. 1992;85:391-405. [Free Full Text]
  6. Smith EB. The relationship between plasma and tissue lipids in human atherosclerosis. Adv Lipid Res. 1974;12:1-49. [Medline] [Order article via Infotrieve]
  7. Stary HC. Evolution and progression of atherosclerotic lesions in coronary arteries of children and young adults. Arteriosclerosis. 1989;9(suppl I):I-19-I-32.
  8. Mitchinson MJ, Ball RY. Macrophages and atherosclerosis. Lancet. 1987;2:146-148. [Medline] [Order article via Infotrieve]
  9. Richardson PD, Davies MJ, Born GVR. Influence of plaque configuration and stress distribution on fissuring of coronary atherosclerotic plaques. Lancet. 1989;2:941-944. [Medline] [Order article via Infotrieve]
  10. Pollak OJ. Mast cells in the circulatory system of man. Circulation. 1957;16:1084-1089. [Medline] [Order article via Infotrieve]
  11. Pouchlev A, Youroukova Z, Kiprov D. A study of changes in the number of mast cells in the human arterial wall during the stages of development of atherosclerosis. J Atheroscler Res. 1966;6:342-351. [Medline] [Order article via Infotrieve]
  12. Kamio A, Taguchi T, Shiraishi M, Shimata K, Takebayashi S, Cleveland JC, Kummerow FA. Mast cells in human aorta. Arterial Wall. 1979;5:125-136.
  13. Trillo AA. The cell population of aortic fatty streaks in African green monkeys with special reference to granulocytic cells: an ultrastructural study. Atherosclerosis. 1982;43:259-275. [Medline] [Order article via Infotrieve]
  14. Stary HC. The sequence of cell and matrix changes in atherosclerotic lesions of coronary arteries in the first forty years of life. Eur Heart J. 1990;11(suppl E):3-19.
  15. 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]
  16. 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]
  17. 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]
  18. Kokkonen JO, Kovanen PT. The metabolism of low density lipoproteins by rat serosal mast cells. Eur Heart J. 1990;11(suppl E):134-146.
  19. 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]
  20. 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.
  21. Lee M, Lindstedt LK, Kovanen PT. Mast cell–mediated inhibition of reverse cholesterol transport. Arterioscler Thromb. 1992;12:1329-1335. [Abstract/Free Full Text]
  22. Ishizaka T, Ishizaka K. Activation of mast cells for mediator release through IgE receptors. Prog Allergy. 1984;34:188-235. [Medline] [Order article via Infotrieve]
  23. Mota I. The mechanism of anaphylaxis, I: production and biological properties of `mast cell sensitizing' antibody. Immunology. 1964;7:681-699.
  24. Martin U, Römer D. The pharmacological properties of a new, orally active antianaphylactic compound: Ketotifen, a benzocycloheptathiophene. Drug Res. 1978;28:770-782. [Medline] [Order article via Infotrieve]
  25. 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]
  26. Pruzansky JJ, Grammer LC, Patterson R, Roberts M. Dissociation of IgE from receptors on human basophils, I: enhanced passive sensitization for histamine release. J Immunol. 1983;131:1949-1953. [Abstract]
  27. 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]
  28. 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]
  29. 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]
  30. Robenek H, Schmitz G, Assman 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]
  31. 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]
  32. Finbloom DS, Metzger H. Binding of immunoglobulin E to the receptor on rat peritoneal macrophages. J Immunol. 1982;129:2004-2008. [Abstract]
  33. Kokkonen JO, Kovanen PT. Low-density-lipoprotein binding by mast-cell granules. Biochem J. 1987;241:583-589. [Medline] [Order article via Infotrieve]
  34. 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]
  35. Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci U S A. 1979;76:333-337. [Abstract/Free Full Text]
  36. 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]
  37. 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]
  38. 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]
  39. Joris I, Majno G. Inflammatory components of atherosclerosis. Adv Inflamm Res. 1979;1:71-85.
  40. Libby P, Hansson GK. Involvement of the human immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5-15. [Medline] [Order article via Infotrieve]
  41. Liu MC, Proud D, Lichtenstein LM, MacGlashan DW, Schleimer RP, Adkinson NF, Kagey-Sobotka A, Schulman ES, Plaut M. Human lung macrophage-derived histamine-releasing activity is due to IgE-dependent factors. J Immunol. 1986;136:2588-2595. [Abstract]
  42. Kaplan AP, Reddigari S, Baeza M, Kuna P. Histamine releasing factors and cytokine-dependent activation of basophils and mast cells. Adv Immunol. 1991;50:237-260. [Medline] [Order article via Infotrieve]
  43. Hogan MB, Patterson R. Heterogeneity of histamine releasing factors and IgE. Allergy Proc. 1993;14:351-355. [Medline] [Order article via Infotrieve]
  44. Mitchinson MJ, Ball RY. Macrophages and atherogenesis. Lancet. 1987;2:146-148.
  45. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488-500. [Medline] [Order article via Infotrieve]
  46. Blaes N, Crouzet B, Bourdillon M, Boissel J. Comparative phagocytosis in culture of aortic smooth muscle cells and fibroblasts from rat. Proc Soc Exp Biol Med. 1982;170:453-458. [Medline] [Order article via Infotrieve]
  47. Wolfbauer G, Glick JM, Minor LK, Rothblat GH. Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions. Proc Natl Acad Sci U S A. 1986;83:7760-7764. [Abstract/Free Full Text]
  48. Galli SJ. New concepts about the mast cell. N Engl J Med. 1993;328:257-265. [Free Full Text]
  49. Sutton BJ, Gould HJ. The human IgE network. Nature. 1993;366:421-428. [Medline] [Order article via Infotrieve]
  50. Tada T, Ishizaka K. Distribution of gamma E-forming cells in lymphoid tissues of the human and monkey. J Immunol. 1970;104:377-387. [Abstract/Free Full Text]
  51. Huggins KG, Brostoff J. Local production of specific IgE antibodies in allergic-rhinitis patients with negative skin tests. Lancet. 1975;1:148-150.
  52. Owndy DR. Clinical significance of IgE. In: Middleton E Jr, Reed CE, Ellis EF, Adkinson NF Jr, Yunginger JW, Busse WW, eds. Allergy: Principles and Practice. St Louis, Mo: Mosby; 1993:1059-1076.
  53. Bernstein ID, Ovary Z. Absorption of antigens from the gastrointestinal tract. Int Arch Allergy. 1968;33:521-527.
  54. Warshaw AL, Walker WA, Isselbacher KJ. Protein uptake by the intestine: evidence for absorption of intact macromolecules. Gastroenterology. 1974;66:987-992. [Medline] [Order article via Infotrieve]
  55. Sampson HA. Role of immediate food hypersensitivity in the pathogenesis of atopic dermatitis. J Allergy Clin Immunol. 1983;71:473-480. [Medline] [Order article via Infotrieve]
  56. Sampson HA, Metcalfe DD. Food allergies. JAMA. 1992;268:2840-2844. [Medline] [Order article via Infotrieve]
  57. Walzer M, Brooklyn NY. Allergy of the abdominal organs. J Lab Clin Med. 1941;26:1867-1877.
  58. Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A. 1991;88:8154-8158. [Abstract/Free Full Text]
  59. Bonfiglio TA, Botti RE, Hagstrom JWC. Coronary arteritis, occlusion, and myocardial infarction due to lupus erythematosus. Am Heart J. 1972;83:153-158. [Medline] [Order article via Infotrieve]
  60. Tsakraklides VG, Blieden LC, Edwards JE. Coronary atherosclerosis and myocardial infarction associated with systemic lupus erythematosus. Am Heart J. 1974;87:637-641. [Medline] [Order article via Infotrieve]
  61. Willerson JT, Guthrie AM, Buja LM. Chest pain in a 26-year-old woman with a history of systemic lupus erythematosus and hypertension. Circulation. 1993;88:787-796. [Free Full Text]
  62. Minick CR. Immunologic arterial injury in atherogenesis. Ann N Y Acad Sci. 1976;275:210-227.
  63. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
H. Ma and P. T. Kovanen
Inhibition of Mast Cell-Dependent Conversion of Cultured Macrophages Into Foam Cells With Antiallergic Drugs
Arterioscler. Thromb. Vasc. Biol., December 1, 2000; 20 (12): e134 - e142.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
Y. Wang and P. T. Kovanen
Heparin Proteoglycans Released From Rat Serosal Mast Cells Inhibit Proliferation of Rat Aortic Smooth Muscle Cells in Culture
Circ. Res., January 22, 1999; 84(1): 74 - 83.
[Abstract] [Full Text] [PDF]

Home page
 Arch Intern MedHome page
P. T. Kovanen, M. Manttari, T. Palosuo, V. Manninen, and K. Aho
Prediction of Myocardial Infarction in Dyslipidemic Men by Elevated Levels of Immunoglobulin Classes A, E, and G, but Not M
Arch Intern Med, July 13, 1998; 158(13): 1434 - 1439.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
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 HighWire
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.

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1995 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.