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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2047-2054.)
© 1995 American Heart Association, Inc.

Articles

Extracellular Mast Cell Granules Carry Apolipoprotein B-100–Containing Lipoproteins Into Phagocytes in Human Arterial Intima

Functional Coupling of Exocytosis and Phagocytosis in Neighboring Cells

Maija Kaartinen; Antti Penttilä; Petri T. Kovanen

From the Wihuri Research Institute (M.K., P.T.K) and the Department of Forensic Medicine, University of Helsinki (A.P.), Helsinki, Finland.

Correspondence to Petri T. Kovanen, MD, Wihuri Research Institute, Kalliolinnantie 4, 00140 Helsinki, Finland.

	Abstract

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Abstract In experimental studies in vitro, mast cells have induced uptake of apolipoprotein B-100 (apoB-100)–containing low-density lipoproteins by macrophages, with the subsequent formation of foam cells, the hallmarks of atherosclerosis. Recently, increased numbers of activated, ie, degranulated, mast cells were found to be present in human coronary fatty streaks and atheromas. We therefore sought evidence of a connection between mast cells and foam cell formation in vivo. In electron microscopic studies of human aortic and coronary fatty streaks and atheromas, exocytosed cytoplasmic secretory granules of mast cells were detected in the vicinity of their parent cells. These exocytosed granules had bound apoB-100–containing lipoproteins, as indicated by their positive staining with MB 47, a monoclonal antibody against apoB-100. A smooth muscle cell was observed to be in the process of phagocytosing one such exocytosed granule, and in the vicinity of a degranulated mast cell a foam cell contained an ingested mast cell granule. Therefore, the micrographs show that exocytosed granules of intimal mast cells may contribute to intimal foam cell formation and suggest a role for mast cells in human atherogenesis. More generally, the findings provide evidence that phagocytosis of apoB-100–carrying particles is one mechanism by which lipoproteins enter human arterial intimal cells.

Key Words: atherosclerosis • exocytosis • mast cells • low-density lipoproteins • phagocytosis

	Introduction

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The earliest recognizable gross lesion in atherogenesis, the fatty streak, is characterized by accumulation of LDL-derived cholesteryl esters in the cells of the arterial intima, with formation of foam cells.^{1 2} Foam cells are also present in the growing edges, the shoulder regions of late atherosclerotic lesions referred to as atheromas. Most foam cells have been shown to arise from macrophages that originally migrated from the circulation,³ and a variable fraction of them are derived from smooth muscle cells that have migrated to the intima from the medial layer of the artery.⁴ Mast cells are also present in the arterial intima, and the number of activated, ie, degranulated, mast cells is increased in human aortic and coronary fatty streaks and in the shoulder regions of atheromas.^{5 6} Moreover, mast cells are an integral part of aortic fatty streaks in cholesterol-fed African green monkeys.⁷

Since uptake of LDL through the classical LDL receptor pathway does not lead to cholesterol accumulation in cells,⁸ foam cell formation must depend on other mechanisms. Indeed, a specific chemical modification of the apoB-100 component of LDL is followed by massive uptake of LDL by another receptor-mediated mechanism, called the "scavenger receptor" pathway.⁹ In addition, foam cells may be generated by phagocytic uptake of LDL aggregates or of LDL bound to other macromolecules or to their aggregates.¹⁰ It is generally held that modifications of LDL and formation of complexes between LDL and macromolecules or their aggregates occur locally in the arterial intima by the action of the intimal cells.¹¹

In previous studies in vitro with rat serosal mast cells, we found that stimulated mast cells induced uptake of LDL by cocultured mouse peritoneal macrophages.¹² In rat serosal mast cells, the model used in our studies, the cytoplasm is filled with secretory granules, which upon stimulation of the mast cells are expelled into the extracellular fluid.^{13 14} There the soluble components of the granules, ie, histamine and a fraction of their proteoglycans, are released and diffuse away. In contrast, the major granule components, two neutral proteases (a chymotryptic endopeptidase chymase and an exopeptidase carboxypeptidase A), and the major fraction of the heparin proteoglycans remain tightly bound to each other, forming extracellular "granule remnants."¹² When rat serosal mast cells were cocultured with mouse peritoneal macrophages and stimulated in the presence of LDL, the apoB-100 component of the LDL particles was bound by the heparin component of the granule remnants, and these LDL-coated granule remnants were phagocytosed by the macrophages, the result being massive uptake of LDL by the macrophages.¹⁵

On stimulation of rat serosal mast cells, most of the granules exposed to the extracellular fluid remain in the degranulation channels, and, like the granule remnants expelled into the extracellular space, these remnants too bind LDL.¹⁶ Such LDL is rapidly (within minutes) internalized by the mast cells, along with the remnants. The mast cells recover rapidly from stimulation and are soon (within hours) ready for a second stimulation. On restimulation, the already–LDL-loaded granule remnants may be expelled and then carry their cholesterol load to macrophages. However, some of these LDL-loaded remnants remain in the channels and bind more LDL. Thus, a mechanism was discovered by which LDL cholesterol can accumulate in mast cells.¹⁶

The presence of activated mast cells in the human arterial intima, where high LDL concentrations prevail and where foam cells are formed, prompted us to search for evidence of mast cell–dependent foam cell formation in this tissue site. The availability of monoclonal antibodies against apoB-100 and against tryptase, a neutral protease specific to human mast cells,¹⁷ allowed us to test this possibility with the aid of immunoelectron microscopic studies. The results revealed a novel extracellular carrier system of LDL in vivo, in which exocytosis and phagocytosis of mast cell granules are coupled. This pathway leads to transcellular transfer of mast cell granule remnants, which thus, when carrying LDL, contributes to the initiation of human atherogenesis.

	Methods

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An autopsy series comprising 19 subjects (15 males, 4 females) 24 to 84 years old was used for this study. The causes of death were cardiovascular disease (n=5), violent deaths, ie, accidents and suicides (n=8), and poisoning with self-administered alcohol and drugs (n=6). The autopsy material (abdominal aorta and left coronary artery) was obtained within 24 hours of death (range 2.5 to 24 hours; mean 10 hours). The aortic samples (n=19) included macroscopically identifiable fatty streaks (n=18) and raised atheromatous lesions (n=15), and the coronary samples included the (unopened) bifurcation area of the left coronary artery (n=10). Specimens for electron microscopy (11 aortic and 5 coronary) were fixed for 3 hours in a solution containing 2% glutaraldehyde and 1.5% paraformaldehyde and postfixed in osmium tetroxide. The samples were then dehydrated, embedded in Epon, sectioned with an ultramicrotome, and finally stained with uranyl acetate and lead citrate. For immunoelectron microscopy, specimens (8 aortic and 5 coronary) were fixed for 3 hours in 3% paraformaldehyde, dehydrated, and embedded in LR white resin. The ultramicrotome sections on nickel grids were incubated at room temperature with buffer (PBS containing 3% BSA) for 10 minutes and then for 4 hours with anti-tryptase G3 monoclonal antibody (kindly provided by Dr L. B. Schwartz, Medical College of Virginia, Richmond) or with anti–apoB-100 MB 47 (1:100) (kindly provided by Dr J. L. Witztum, University of California, San Diego, La Jolla SCOR). After two rinses in the buffer, the sections were incubated for 2 hours in 10 nm gold-labeled anti-mouse IgG+IgM (1:50) (Amersham) and finally stained with uranyl acetate and lead citrate. In sections serving as negative controls, the primary antibody was omitted or replaced with normal serum (irrelevant antibody). In these sections no apoB-100 staining was observed. The sections (1 µm) were stained with toluidine blue and observed with light microscopy for orientation of the site and for evaluation of the atherosclerotic involvement of the arteries.^{5 6} The ultramicrotome sections were viewed with a Jeol JEM-1200EX transmission electron microscope at the Department of Electron Microscopy, University of Helsinki.

	Results

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Fig 1 shows a light micrograph of a section of an atherosclerotic human left coronary artery stained with toluidine blue. The lesion was classified as a fatty streak because it contained foam cells. This lesion also contained typical mast cells, which could be identified by their contents of metachromatic cytoplasmic granules. We could identify mast cells in all coronary fatty streaks examined (number of subjects, 10).

View larger version (28K): [in this window] [in a new window]   Figure 1. Light microscopic view of a section of an atherosclerotic human left coronary artery. The lesion is classified as a fatty streak because it contains foam cells (a typical foam cell is indicated by the arrowhead). The lesion also contains mast cells (arrows) with typical metachromatic cytoplasmic granules. The specimen was from a 41-year-old man who died of aortic dissection. The section is stained with toluidine blue; magnification x200.

To search for evidence of the granule carrier mechanism in which apoB-100–containing lipoproteins are bound to exocytosed mast cell granules, we turned to immunoelectron microscopy. For this purpose, coronary fatty streaks from five subjects were studied. First, mast cell granules were identified with anti-tryptase antibody. Fig 2 shows an immunoelectron micrograph of a mast cell present in a fatty streak lesion of the coronary intima. Several intracellular tryptase-positive granules and four tryptase-positive extracellularly located mast cell granule remnants are visible. Indeed, 138 (71%) of the total of 194 mast cells that were identified and photographed had extracellular granule remnants in their vicinity. Therefore, in the atherosclerotic lesion shown (as in the other lesions studied), mast cells were degranulated, signifying their stimulation. Most important, the necessary condition for the granule carrier mechanism to operate, ie, the presence of granule remnants in the extracellular space, was fulfilled.

View larger version (185K): [in this window] [in a new window]   Figure 2. Electron micrograph showing an activated mast cell in a coronary fatty streak lesion. The section is stained with anti-tryptase antibody, which is detected with 10-nm gold-labeled IgG+IgM as secondary antibodies. Several tryptase-positive cytoplasmic secretory granules within the cell and four extracellularly located granule remnants (arrows) are visible. The specimen was from a 42-year-old man who died of poisoning with self-administered alcohol. Bar=0.2 µm.

We next searched for evidence of apoB-100 binding to mast cell granule remnants and stained the sections with monoclonal antibody MB 47 against the apoB-100 component of lipoproteins. Fig 3 shows an immunoelectron micrograph of a stimulated mast cell with one exocytosed granule. This extracellularly located granule remnant stained for MB 47, an observation made in three of the five samples studied. In sharp contrast, the extracellular matrix seen in this specimen is totally devoid of apoB-100, as evidenced by failure to stain for MB 47. In the other samples studied, occasional gold particles could be observed in the extracellular matrix; in each case, however, the density of the gold particles was smaller in the matrix than in the extracellular mast cell granules.

View larger version (146K): [in this window] [in a new window]   Figure 3. Demonstration of apoB-100 binding to mast cell granules. The sections were stained with MB 47, a monoclonal antibody against apolipoprotein B-100 (apoB-100). The primary antibody is detected with 10-nm gold-labeled IgG+IgM as secondary antibodies. A degranulated mast cell (left) with an exocytosed granule (arrow) that stains positively for MB 47 is shown. Note the opening of a degranulation channel into the extracellular space (between arrowheads) and some apoB-100 in the path of the exocytosed granule. The rest of the extracellular area is totally devoid of apoB-100. The specimen was from a 66-year-old man who died of myocardial infarction. Bar=0.2 µm.

As described above, the granule remnants remaining in the degranulation channels of rat serosal mast cells bind LDL particles and internalize them. Similarly, in the mast cells of the coronary lesions we regularly found apoB-100–positive granule remnants (ie, the granules had bound LDL or other apoB-100–containing lipoproteins), whereas the cytoplasm of such cells was practically devoid of apoB-100. Fig 4 shows an immunoelectron micrograph of a mast cell with several intracellular granules that stain positively for MB 47. Notably, the MB 47–positive granules are electron transparent and the MB 47–negative granules are electron dense. Electron transparency of a granule reveals that the particular granule has been in contact with the extracellular fluid and has lost some of its contents.^{16 18} The above finding was made in four of the five samples studied and corroborates the idea that any apoB-100 bound to intracellular granule remnants has an extracellular origin.

View larger version (153K): [in this window] [in a new window]   Figure 4. Demonstration of apolipoprotein B-100 (apoB-100) inside a mast cell. The sections were stained with MB 47, a monoclonal antibody against apoB-100. The primary antibody is detected with 10-nm gold-labeled IgG+IgM as secondary antibodies. A mast cell (top) contains several intracellular granules (arrows) that stain positively for MB 47. Notably, the MB 47–positive granules are electron transparent and the MB 47–negative granules (arrowheads) are electron dense. The specimen was from a 66-year-old man who died of myocardial infarction. Bar=1 µm.

Fig 5A shows an electron micrograph of a mast cell and a smooth muscle cell in an aortic fatty streak. In addition to the electron-dense cytoplasmic granules, electron-transparent granules and also empty membrane-bound vacuoles can be seen inside the mast cell, revealing that this particular cell has previously been stimulated to degranulate. At higher magnification (Fig 5B), one exocytosed mast cell granule (arrow) can be detected. At this magnification, the heterogeneous morphology of the mast cell granules can be discerned: Several intracellular granules contain the scrolls¹⁸ typical of human mast cells (seen in both cross and longitudinal sections). The single extracellularly located granule also displayed the typical structural pattern of mast cell granules, ie, several sets of parallel stripes (scrolls in longitudinal view) and one set of concentric circles (scroll in cross section; arrow). This panel also shows a large cytoplasmic cell projection (pseudopod) extending from the smooth muscle cell at the site where the granule remnant is located. Fig 5C shows the next section cut from this area, in which the remnant is surrounded by two smaller pseudopods, one on each side. Thus, the smooth muscle cell appears to be in the process of engulfing the granule remnant. This set of micrographs displayed all the necessary components to perform the steps of the granule-mediated carrier pathway (ie, the parent mast cell, the exocytosed granule remnant, and the phagocytosing cell with pseudopods directed toward the granule remnant) visualized together. This finding was observed in 2 of 18 aortic samples.

View larger version (542K): [in this window] [in a new window]   Figure 5. (This page and previous page). An exocytosed mast cell granule about to be engulfed by a smooth muscle cell. A, A mast cell (left) containing several granules of varying appearance (electron dense and electron lucent) is located near a smooth muscle cell (right). Bar=2 µm. B, Detailed view of the mast cell (MC), the smooth muscle cell (SMC), and the extracellular space between the mast cell and the smooth muscle cell. Inside the mast cell secretory granules of varying morphology (scrolls in cross section [arrow], scrolls in longitudinal section [arrowhead], and dense granular contents [open arrow]) are seen. Note that the smooth muscle cell has extended a large pseudopod (*) next to the extracellular granule remnant (arrow). Bar=0.2 µm. C, Section adjacent to A and B (at 60 nm distance) showing two small cytoplasmic projections (arrows), one on each side of the granule remnant, extending from the larger pseudopod (*). The smooth muscle cell appears to be in the process of engulfing the granule remnant. Bar=0.2 µm. The specimen was from a 63-year-old man who died of myocardial infarction.

Fig 6 shows an immunoelectron micrograph of an aortic fatty streak in which mast cell granules were identified with the anti-tryptase antibody. Adjacent to a mast cell, a small section of a typical foam cell with large empty vacuoles is seen. The foam cell contains one tryptase-positive particle that is thus identified as a granule remnant. In three of eight aortic samples studied, a granule remnant–containing foam cell could be observed.

View larger version (163K): [in this window] [in a new window]   Figure 6. Demonstration of a mast cell granule inside a foam cell. A foam cell (right) can be identified near a mast cell (left) (cell boundaries are marked with arrowheads). The foam cell (with two cytoplasmic vacuoles [*]) contains a small cytoplasmic inclusion, which, being tryptase- positive, can be identified as a mast cell granule (arrow). The section was stained with anti-tryptase antibody, which is detected with 10-nm gold-labeled IgG+IgM as secondary antibodies. The specimen was from a 51-year-old man who died of poisoning with self-administered alcohol. Bar=1 µm.

	Discussion

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The current study demonstrates that exocytosed mast cell granules, ie, granule remnants, bind apoB-100–containing lipoproteins present in the intimal fluid of human aortic and coronary vessels. Indeed, we regularly observed binding of apoB-100 to the exocytosed granules. Since some of the apoB-100–containing lipoproteins are lost during tissue processing (fixation, dehydration, and staining), the above observation shows that the apoB-100–containing lipoproteins are bound to the exocytosed granules rather than to the extracellular matrix. This suggestion is supported by the fact that, among the glycosaminoglycans present in the intima, heparin, the component of mast cell granules responsible for binding apoB-100, has the highest density of negative charges.¹⁹ Heparin can therefore interact most extensively with the positively charged amino acid residues of the apoB-100.²⁰ Another reason for the more intense reaction of MB 47 with LDL bound to the heparin proteoglycans of granule remnants than with LDL bound to (chondroitin sulfate) proteoglycans of the arterial extracellular matrix could be differences in binding of apoB-100 to these two types of proteoglycans,^{21 22 23} thus leading to differential exposure (or steric hindrance) of the epitope responsible for binding MB 47.²⁴ Moreover, a specific mechanism that increases the strength of binding between LDL particles and mast cell granule remnants was recently discovered.²⁵ When the apoB-100 component of granule-bound LDL is proteolyzed by granule chymase, the LDL particles become unstable and fuse, and the fused particles bind more tightly to the granule remnant heparin proteoglycans.

The present study also describes a mechanism by which exocytosed mast cell granules carry apoB-100–containing lipoproteins into intimal cells. The electron microscopic studies do not allow us to conclude which of the apoB-100–containing lipoproteins present in the intimal fluid [VLDL remnant–like particles, LDL, or LP(a)]^{26 27 28} had actually bound to the granules. Since, of the total immunoreactive apoB-100 in lipoproteins isolated from atherosclerotic intima, most (>95%) is present in LDL-like particles, it is likely that the majority of granule-associated apoB-100 also represents binding of LDL.^{26 29}

The efficiency of the granule-remnant carrier system in the intima must depend on the number of carriers available and their binding capacity. In atherosclerotic lesions the number of degranulated mast cells is increased,⁶ indicating that during atherogenesis more granules are available for lipoprotein binding. The binding capacity of exocytosed rat mast cell granules has been studied in detail.³⁰ Thus, each exocytosed granule has the capacity, depending on its size, to bind maximally 5000 to 10 000 LDL particles. We have also observed that the proteolytic activity of a granule remnant increases its maximal capacity to bind LDL.³¹ Degradation of the apoB-100 moiety of the granule-remnant–bound LDL by the also granule-remnant–bound chymase is followed by fusion of LDL particles, whereupon the capacity of the remnants to carry LDL increases fivefold. Accordingly, the human granule remnants containing chymase in addition to tryptase have the potential to bind extra LDL and thus become even more efficient carriers. Since the concentration of LDL usually found in the intimal fluid²⁷ (1500 µg/mL of apoB-100) is far higher than that required to saturate granule-remnant binding sites (25 µg/mL), the exocytosed granules in the intimal fluid are likely to be maximally loaded with LDL. However, the current morphological studies do not allow any conclusions about the quantitative significance of the granule carrier mechanism in foam cell formation in human arterial intima. Neither have other mechanisms of foam cell formation been quantified in human atherogenesis. Thus, it remains to be shown how important the newly described mechanism is in comparison with all of the other mechanisms of intimal foam cell formation.

The natural fate of an exocytosed mast cell granule is to be phagocytosed by the cells present in the vicinity of the degranulated mast cell.^{32 33} Hence, the factors responsible for granule-remnant traffic in the intimal space are the rates of granule exocytosis and granule phagocytosis. The mast cells in the atherosclerotic areas reside in an inflamed area where many potential mast cell activators are present. Among these are activated T lymphocytes, activated macrophages, and activated complement.³⁴ Indeed, in human coronary fatty streaks the degree of mast cell degranulation was recently found to be markedly increased (from 18% in normal intima to 52% in fatty streaks).⁶ Similarly, in the present study 71% of the mast cells in the arterial (coronary or aortic) intima showed signs of degranulation. Thus, we can infer that the majority of the mast cells actively participated in the granule-remnant carrier system. In contrast to degranulation (ie, granule exocytosis), no quantitative data are available on granule phagocytosis in the various intimal lesions. However, it appears that, in the intima, mast cells reside in a tissue composed of many cells capable of phagocytosis. Thus, as the smooth muscle cells leave the medial layer and enter the intima, their phenotype is switched from contractile to synthetic, and this change in phenotype is accompanied by an apparent increase in phagocytotic capacity.³⁵ Indeed, we recently found that smooth muscle cells of synthetic phenotype actively phagocytose exocytosed mast cell granules, and that, in vitro, the granule remnants can carry LDL into the cells and induce their conversion into foam cells.³⁶ Similarly, as the blood monocytes enter the atherosclerotic intima, they differentiate into tissue macrophages and become activated, and their phagocytic capacity increases.³⁷ Accordingly, the necessary conditions for the two events in the granule carrier pathway, granule exocytosis and phagocytosis, prevail in the atherosclerosis-prone areas of human arterial intima.

A carrier mechanism for cellular uptake of LDL was originally described in a cell culture system in which formation of LDL–dextran sulfate complexes in the incubation medium was associated with uptake of cholesteryl esters by mouse macrophages ("piggyback system").³⁸ Later, uptake of complexes between LDL and proteoglycans obtained from the arterial wall was also described.^{39 40 41} However, phagosomal uptake of such complexes in human atherosclerotic lesions has eluded direct observation so far. The current observations provide the first visual evidence that an exocytosis-phagocytosis–coupled carrier mechanism leads to uptake of apoB-100–containing lipoproteins by phagocytes in the human arterial intima. Unraveling the factors that regulate this novel pathway in the arterial intima is a challenge for the future.

	Acknowledgments

 
This study was supported in part by the Aarne Koskelo Foundation and the Meilahti Foundation. The authors thank the staff of the Department of Forensic Medicine and Electron Microscopy, University of Helsinki, for their excellent technical assistance.

Received March 2, 1995; accepted August 14, 1995.

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