Published online before print May 2, 2002, doi: 10.1161/01.RES.0000020017.84398.61
© 2002 American Heart Association, Inc.
Cellular Biology |
Platelet Phagocytosis and Processing of ß-Amyloid Precursor Protein as a Mechanism of Macrophage Activation in Atherosclerosis
From the Division of Pharmacology (G.R.Y.D.M., D.M.M.D.C., D.M.J., W.M., A.G.H., H.B.), University of Antwerp, Antwerp, Belgium; the Department of Anatomical Pathology (S.C.), Electron Microscopy Unit, University of the Orange Free State, Bloemfontein, South Africa; HistoGeneX (M.W.M.K.), Edegem, Belgium; and the Department of Pathology (M.M.K.), General Hospital Middelheim, Antwerp, Belgium.
Correspondence to Guido R.Y. De Meyer, Division of Pharmacology, University of Antwerp (UIA), Universiteitsplein 1, B-2610 Antwerp, Belgium. E-mail gdemeyer{at}uia.ua.ac.be
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Abstract |
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Abstract— In human occluded saphenous vein grafts, we previously demonstrated cytotoxic foam cells, presumably derived from macrophages engulfing platelets. In the present study, we investigated whether platelet phagocytosis occurs in human atherosclerotic plaques, whether this activates macrophages, and whether the platelet constituent, amyloid precursor protein (APP), was involved. Immunohistochemistry documented the presence of APP, ß-amyloid peptide (Aß, cleaved from APP), and platelets (CD9), along with inducible NO synthase (iNOS) and cyclooxygenase-2, two markers of macrophage activation, around microvessels in advanced human carotid artery plaques (n=18). Aß colocalized with iNOS-expressing macrophages that were often surrounded by platelets. In vitro, murine J774 and human THP-1 macrophages were incubated with or without washed human platelets. Coincubation of macrophages and platelets led to platelet phagocytosis (electron and confocal microscopy) and formation of lipid-, APP-, and Aß-containing foam cells. These expressed iNOS mRNA (reverse transcription–polymerase chain reaction) and protein and produced nitrite and tumor necrosis factor-
(ELISA). Macrophage pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride, a protease inhibitor, reduced APP processing and inhibited NO biosynthesis induced by platelet phagocytosis but not by lipopolysaccharides. Human atherosclerotic plaques and J774 and THP-1 macrophages contained mRNA of the APP-cleaving enzyme ß-secretase. This is the first demonstration of Aß, a peptide extensively studied in Alzheimer’s disease, in human atherosclerotic plaques. It was present in activated iNOS-expressing perivascular macrophages that had phagocytized platelets. In vitro studies indicate that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to Aß, resulting in iNOS induction. This represents a novel mechanism for macrophage activation in atherosclerosis.
Key Words: ß-amyloid peptide • amyloid precursor protein • foam cells • inducible nitric oxide synthase • nitric oxide
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Introduction |
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The composition of an atherosclerotic plaque is an important determinant of plaque stability. Unstable rupture-prone plaques are characterized by a thin fibrous cap that contains few smooth muscle cells.1,2 Several lines of evidence suggest that macrophage activation in the vulnerable shoulder of the plaque could contribute to plaque rupture.3,4 We have previously postulated the release of factors toxic to smooth muscle cells, possibly NO, from activated macrophages in human atherosclerotic plaques.5,6 It has been reported that foam cell formation can be induced by platelet phagocytosis.7–10 Moreover, in human (sub)occluded saphenous vein grafts, the formation of toxic foam cells within mural thrombi is presumably the result of platelet phagocytosis by macrophages.11 Therefore, we questioned whether in human atherosclerotic plaques platelet phagocytosis evokes macrophage activation and whether proteolytic processing of amyloid precursor protein (APP), present in platelet
-granules,12–15 is involved in this process. To test this hypothesis, we first documented in human atherosclerotic plaques the presence of APP and ß-amyloid peptide (Aß), which is cleaved from APP and which has been extensively studied in Alzheimer’s disease.16 Furthermore, we investigated the colocalization of APP and Aß with inducible NO synthase (iNOS), a marker of macrophage activation. We then exposed human and murine macrophages in culture to human platelets and demonstrated that platelet phagocytosis resulted in foam cell formation and iNOS induction and that proteolytic processing of APP by macrophages is involved in the latter process.
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Materials and Methods |
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Immunohistochemical Study of Advanced Human Atherosclerotic Plaques
The present study has been approved by the Review Board of the University of Antwerp.
Human carotid endarterectomy specimens were obtained from normocholesterolemic patients (9 men and 9 women, mean age 72±5 years, with carotid stenosis >70%) and processed as previously described.6
For immunohistochemistry, the following antibodies were used: APP (clone 22C11, Roche), Aß (monoclonal antibody, gift from Dr M. Mercken, Janssen Research Foundation, Beerse, Belgium), iNOS (SA-200, Biomol), cyclooxygenase (COX)-2, Transduction Laboratories), and CD9 (gift from Dr F. Lanza, INSERM U. 311, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg, France), an integral membrane protein abundantly present in platelet membranes.17 The Aß antibody specifically stained ß-amyloid deposits in brain tissue from patients with Alzheimer’s disease, confirming its specificity. Double-labeling immunohistochemistry was performed to detect colocalization of iNOS or COX-2 with APP, Aß, or CD9. The iNOS and COX-2 antibodies were detected with Envision (Dako). The APP, Aß, and CD9 antibodies were detected with a horse anti-mouse biotin-labeled secondary antibody (Vector Laboratories). Thereafter, a streptavidin–biotin–alkaline phosphatase complex was formed. Nuclei were stained with hematoxylin.
Cell Culture
The murine macrophage cell line J774A.1 and the human monocyte cell line THP-1 (American Type Culture Collection) were grown in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 150 U/mL polymyxin B, and 50 µg/mL gentamycin. THP-1 cells were differentiated into macrophages by treatment with phorbol 12-myristate 13-acetate (0.2 µmol/L, Sigma Chemical Co). J774 or THP-1 cells (0.5x106/800 µL) were allowed to adhere in culture slides (Becton Dickinson Labware) at 37°C in 5% CO2/95% air. Thereafter, medium was replaced with DMEM supplemented with antibiotics.
The macrophages were incubated for 2, 18, or 41 hours, with or without washed human platelets (108 per 0.5x106 macrophages). Human platelet concentrates (Blood Transfusion Center, University Hospital of Antwerp) had their leukocytes removed by filtration and contained only one to two white blood cells per 300 000 platelets. As a negative control, J774 macrophages were incubated for 41 hours with human white blood cells (up to 3000 white blood cells per 0.5x106 macrophages, which is 
5 to 10-fold more than the potential contamination of the platelet-macrophage incubations).
The responses to platelet phagocytosis were studied in the presence and absence of thrombin (1.25 U/mL, Sigma) and recombinant murine cytokines (100 U/mL interferon-
[IFN-], Sigma; 50 to 100 U/mL interleukin-1ß [IL-1ß], R&D Systems; 50 to 100 ng/mL tumor necrosis factor- [TNF-], Sigma) for J774 cells or a cocktail of recombinant human cytokines (1000 U/mL IFN- , 50 U/mL IL-1ß, and 100 ng/mL TNF-) for THP-1 cells.18,19 The effect of Aß1–40 (50 µmol/L, Bachem) was investigated in J774 cells. Aß1–40 solutions were prepared as described,20 and aggregation of Aß1–40 was confirmed by gel filtration.
In some experiments, the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine HCl (L-NIL, Alexis) was added to the macrophages during the whole incubation period.
To study the effects of an irreversible protease inhibitor with known ß-secretase–inhibiting properties,21 macrophages were pretreated with 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, 0.3 or 0.6 mmol/L, Sigma) for 20 minutes before being seeded on culture slides. This pretreatment had no effect on cell viability. To control for aspecific effects of AEBSF on NO production, lipopolysaccharide (LPS) from Salmonella typhosa (10 µg/mL) was used as a stimulus for iNOS induction. In this case, polymyxin B was omitted from the medium.
NO synthase activity was assessed by measuring nitrite by using the Griess reaction (Schmidt et al22). Mouse TNF- was quantified in the supernatant by using a specific ELISA (Bender Medsystems).
Cytochemistry, Immunocytochemistry, and Scoring
After 41 hours, the cell-free supernatant was stored at -20°C for nitrite determination. The cells were fixed with paraformaldehyde (1%, 2 minutes), followed by methanol (-20°C, 6 minutes), air-dried at room temperature, and stained with oil red O or immunostained for CD9, iNOS, APP, or Aß (see above). The oil red O or APP content of the cells was scored by an independent observer using the following system: 0, negative; 1, cells with only a few positive granules; 2, cells with less than half of the cytoplasm filled with positive granules; 3, cells with more than half of the cytoplasm filled with positive granules; and 4, cytoplasm of the cells completely filled with positive granules.
Electron Microscopy of Cultured Macrophages
Macrophages were incubated with or without platelets for 2 hours. The cells were washed, trypsinized, fixed in a sodium cacodylate (0.15 mol/L)–buffered 1% glutaraldehyde solution for 2 hours, and stored in 0.15 mol/L sodium cacodylate buffer, pH 7.4, until processing. The cells were postfixed in a veronal acetate–buffered 1% OsO4 solution for 1 hour and dehydrated in acetone. Then, they were impregnated with acetone plus Spurr epoxy resin (1:1), subsequently impregnated with pure Spurr epoxy resin, and embedded in BEEM capsules (Better Equipment for Electron Microscopy Inc). Ultrathin sections were stained with uranyl acetate and lead citrate. The macrophages were evaluated in a Philips Technai 10 and Philips 301 transmission electron microscope at 60 kV.
Confocal Microscopy
J774 macrophages and washed human blood platelets were incubated for 45 minutes with DMEM supplemented with 5 µmol/L of 5- and 6-([(4-chloromethyl)benzoyl]amino)tetramethylrhodamine (Cell Tracker Orange, Molecular Probes) or 2.5 µmol/L 5-chloromethylfluorescein diacetate (Cell Tracker Green, Molecular Probes), respectively. Thereafter, media were refreshed, and the macrophages were incubated with the platelets for 2 hours. Dual-channel images were taken with a confocal laser scanning microscope (LSM510, Zeiss). Individual macrophages were isolated from Z stacks with the extract region feature and further analyzed by using the ortho and gallery displays of the LSM510 imaging software.
RNA Extraction and RT-PCR Analysis
Total RNA was isolated23 and reverse transcription (RT)–polymerase chain reaction (PCR) was performed with a 1-step system (Titan, Roche). The following specific primers were used: mouse ß-site APP-cleaving enzyme (BACE) mRNA (which possesses all the known properties of ß-secretase,24 GenBank AF190726), sense 5' TTG CCA GTT GCT TTA GTG ATA 3' and antisense 5' CTT TTT CCC CCA TTT CAT TTC 3'; human BACE mRNA (GenBank AF201468), sense 5' CGG GAG TGG TAT TAT GAG G 3' and antisense 5' GTA TTG CTG CGG AAG GAT G 3'. Human iNOS and ß-actin primers were used as described.25 RT was performed at 50°C for 30 minutes. Thermocycling parameters were as follows: denaturation at 94°C for 2 minutes and 35 cycles consisting of incubations at 94°C for 30 seconds, 55°C for 30 seconds, and 68°C for 45 seconds. In the last 25 cycles, there was an elongation of 5 seconds for each cycle. Finally, a prolonged elongation time of 7 minutes at 68°C was applied. Products were analyzed by agarose gel electrophoresis.
Statistical Analysis
The nitrite values in the different treatment groups were compared by using either the unpaired t test or ANOVA, followed by the Bonferroni test. If the variances were unequal, the data were logarithmically transformed. The scores of oil red O and APP staining were compared by use of the Mann-Whitney test. To study the effect of Aß1–40 on nitrite production by macrophages, 6 batches of Aß1–40 were tested in triplicate. Per batch, the mean nitrite value was calculated, and the responses to Aß1–40 were evaluated pairwise (with versus without Aß1–40) by the Wilcoxon signed rank test.
A value of P<0.05 was considered to be significant. The letter n represents the number of experiments, each of them performed at least in triplicate.
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Results |
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Aß- and iNOS-Expressing Macrophages in Advanced Human Atherosclerotic Plaques
All advanced plaques (n=18) were characterized by foci of neovascularization, inflammatory infiltrates, and a necrotic core (Figure 1A). In all plaques, the regions both adjacent to and distant from the necrotic core contained numerous foam cells of macrophage origin, some of which expressed iNOS (Figures 1A through 1C). APP was present in the cytoplasm of the endothelial cells of neovessels (Figure 1B), which were negative for Aß (Figure 1C). In contrast, the macrophages showed mainly Aß (n=18, Figure 1C) and only a weak APP signal (Figure 1B), suggesting processing of APP within the foam cells. The Aß-immunoreactive macrophages present in the region of neovascularization costained for iNOS (Figure 1C). These macrophages were often surrounded by a rim of immunoreactivity for the platelet membrane protein CD9 (Figure 1D). Macrophages expressing COX-2 often showed colocalization with platelets as well (Figure 1E). Nonatherosclerotic human mammary arteries were negative for APP, Aß, and iNOS (not shown).
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Platelet Phagocytosis Results in the Formation of Lipid-Laden Macrophages
The macrophages that were incubated for 2 or 18 hours with blood platelets clearly showed platelet phagocytosis, as demonstrated by electron microscopy (Figure 2). In these early stages, the phagocytized platelets were still recognizable both by their shape and by the presence of -granules. In addition, confocal laser scanning microscopy of labeled J774 macrophages and platelets confirmed the ingestion of platelets by the macrophages after 2 hours of incubation (Figure 3). Platelet phagocytosis by the macrophages resulted in the formation of lipid-laden cells at 41 hours, as shown with oil red O stain Figures 4A and 4B). The median score of this staining was 0 in the slides without platelets and 3 in the slides with platelets (n=7, P=0.001). J774 macrophages were negative for the platelet membrane protein CD9 (Figure 4C). However, after platelet incubation, the macrophages showed abundant CD9 immunoreactivity (Figure 4D), which was also present in platelets not associated with macrophages.
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Induction of iNOS After Platelet Phagocytosis by Murine and Human Macrophages
Incubation of murine macrophages with platelets resulted in a modest nitrite production. Priming of J774 macrophages with IFN- led to a higher nitrite production and raised the response to platelets enormously (Figure 5A), and this was associated with macrophage cell death. The nitrite formation could be inhibited by the selective iNOS inhibitor L-NIL26 (data not shown). This treatment protected the macrophages from cell death. The biosynthesis of TNF- in response to IFN- and platelets paralleled nitrite production (Figure 5B). The addition of other cytokines, such as mouse TNF- (50 and 100 ng/mL) or mouse IL-1ß (50 and 100 U/mL), neither induced nitrite production nor affected the response to platelets. Platelet activation with thrombin during the incubation did not affect nitrite production (not shown).
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J774 macrophages alone were negative for iNOS protein (Figure 4E) but became clearly immunoreactive after incubation with platelets (Figure 4F). To exclude the possibility that cytokines produced by the few leukocytes contaminating the platelet preparation were responsible for iNOS induction, murine macrophages were also incubated with human leukocytes (up to 3000 per 0.5x106 macrophages). Although this was 5- to 10-fold more than possibly present in the platelet-macrophage incubations, it resulted in neither foam cell formation nor iNOS induction.
Induction of iNOS after platelet phagocytosis was confirmed with human macrophages. Unstimulated THP-1 cells did not contain iNOS mRNA or protein, but after incubation with platelets, both mRNA and protein were expressed in the macrophages (Figures 6A through 6C). Furthermore, incubation of THP-1 macrophages with platelets led to stimulation of nitrite production (Figure 6D). The stimulation was equivalent to the effect of a human recombinant cytokine cocktail (1000 U/mL IFN-, 100 ng/mL TNF-, and 50 U/mL IL-1ß). The combination of this cocktail and platelets resulted in an additive (not synergistic) increase in nitrite production (not shown).
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Stimulation of J744 Macrophages With Aß1–40 Leads to Enhanced Nitrite Production
Five of 6 batches of Aß1–40 increased nitrite production but only in the presence of IFN- (500 U/mL).27,28 The mean nitrite values were 0.71 µmol/L in unstimulated macrophages, 0.64 µmol/L in Aß1–40-treated macrophages, 1.41 µmol/L in IFN-–stimulated macrophages, and 2.03 µmol/L in macrophages stimulated with Aß1–40 and IFN- (P<0.05 versus IFN- alone, Wilcoxon signed rank test, n=6).
AEBSF Reduces APP Processing and NO Formation After Platelet Phagocytosis
J774 macrophages were negative for APP (Figure 4G), whereas human platelets showed a strong immunoreactivity (Figure 4H). Incubation of macrophages with platelets resulted in the occurrence of a granular cytoplasmic APP immunoreactivity, compatible with the uptake of platelets (Figure 4H and online Figure, which can be found in the online data supplement available at http://www.circresaha. org). A brief pretreatment of the J774 macrophages with the irreversible protease inhibitor AEBSF increased the cytoplasmic APP signal in the macrophages (Figures 7A and 7B; median score 2, n=16) compared with nontreated macrophages (median score 1, n=19; P=0.001). After platelet incubation, the oil red O stain of AEBSF-pretreated macrophages did not differ from that of nontreated macrophages (not shown).
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Pretreatment of the macrophages with AEBSF concentration-dependently reduced nitrite production of IFN-–primed macrophages incubated with platelets, whereas their response to LPS was not affected (Figure 7C). Pretreatment of the platelets with AEBSF was without effect (data not shown).
Macrophages Contain Aß After Platelet Phagocytosis and Possess ß-Secretase mRNA
J774 macrophages incubated with platelets (Figure 4J) showed an abundant immunoreactivity for Aß compared with control macrophages (Figure 4I). Immunoreactivity for Aß could not be detected in free platelets not associated with macrophages. RT-PCR analysis confirmed that the mRNA of BACE, which possesses all the known characteristics of ß-secretase,24 was present both in human atherosclerotic plaques and in J774 and THP-1 macrophages (Figure 8). Omitting either the RNA or the RT step did not result in a band on the gel.
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Discussion |
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Hitherto, APP and Aß have been almost exclusively investigated in brain tissue in Alzheimer’s disease. In the present study, we demonstrated for the first time that both APP and Aß were present in advanced human atherosclerotic plaques, more specifically in macrophages associated with platelet phagocytosis in the vicinity of neovascularization. Although endothelial cells of plaque microvessels also contained APP, as reported for neovessels in thrombi,29 they did not show strong Aß immunoreactivity, in contrast to the perivascular macrophages. Another new finding was that platelet phagocytosis and the presence of Aß were associated with macrophage activation, as indicated by the colocalization with iNOS and COX-2 expression in perivascular foam cells. To investigate whether platelet phagocytosis evokes macrophage activation and the possible role of APP and Aß in this process, cell culture experiments were performed with the use of iNOS activity and biosynthesis of TNF-
as markers of macrophage activation.
Phagocytosis of platelets by J774 macrophages led to the formation of foam cells, as previously described.7–10 An interesting new finding was that incubation of IFN-–primed macrophages with human blood platelets led to macrophage activation, as indicated by the production of high amounts of nitrite and TNF-. The nitrite production was iNOS-mediated, inasmuch as it could be inhibited by the selective iNOS inhibitor L-NIL and was not due to contamination of the platelets with LPS, because the incubations were performed in the presence of polymyxin B, which binds possible traces of LPS.30 Contamination of the platelets with white blood cells could also be excluded as a possible stimulus for iNOS expression. On the other hand, priming of the macrophages with IFN- was a prerequisite to obtain large-scale iNOS induction. In advanced human atherosclerotic plaques, many T lymphocytes are present31 (see also Figure 1), which could be a possible source of IFN-.
In human macrophages, it is extremely difficult to induce nitrite formation in vitro, and the classic IFN-–LPS mixture is never successful, as reported by Jorens et al32 and Albina.33 However, in the present study, we demonstrate that human THP-1 macrophages significantly increase nitrite production after platelet incubation. Although nitrite levels were low compared with the murine system, it is important to note that they were equivalent to those obtained with the most active stimuli reported in the literature.33 Furthermore, platelet exposure alone was sufficient to induce iNOS mRNA and protein. Taken together, our in vitro findings fitted with the observation (in human atherosclerotic plaques) that platelet phagocytosis was associated with iNOS expression.
Having demonstrated that platelet phagocytosis leads to macrophage activation, we then examined whether platelet-derived APP was involved. Although J774 macrophages express high levels of iNOS and produce large amounts of NO when they are cultured with IFN- in the presence of LPS, zymosan, Staphylococcus aureus, or Leishmania major, phagocytosis per se is not sufficient for the induction of iNOS in macrophages.34 In IFN-–primed microglia and macrophages, Aß has been reported to induce iNOS expression.20,27,28 Because platelets contain APP in their -granules,12–15 we investigated the hypothesis that platelet phagocytosis evokes macrophage activation via proteolytic processing of platelet-derived APP, similar to APP processing in microglia in brain tissue. First, we showed that the mRNA of BACE, an APP-cleaving enzyme,24 is expressed in human atherosclerotic plaques and macrophages. A brief pretreatment with AEBSF, an irreversible protease inhibitor that inhibits BACE,21 reduced APP processing and inhibited nitrite formation from IFN-–stimulated macrophages in response to platelets but not to LPS, indicating that the inhibitory effect of AEBSF was not due to interference with the cell signaling pathways essential for iNOS induction. AEBSF did not inhibit platelet phagocytosis, inasmuch as oil red O staining of AEBSF-pretreated macrophages did not differ from that of untreated macrophages (not shown). Pretreatment of platelets with AEBSF did not influence nitrite production, indicating that APP processing did not occur in the platelet. Collectively, these data support the hypothesis that proteolytic processing of platelet-derived APP by macrophages is involved in macrophage activation after platelet phagocytosis. However, besides BACE, other proteolytic enzymes, such as caspases,35,36 could be involved in the proteolytic processing of platelet APP as well. Finally, we showed that Aß1–40, a peptide derived from APP by the action of BACE, activated J774 macrophages to produce nitrite. Aß1–40 activation of J774 macrophages required the presence of IFN-, which confirmed the findings of previous studies.27,28 Furthermore, this activation was also analogous to the response to platelets. Although 5 of 6 batches of Aß1–40 tested in triplicate increased nitrite production in the presence of IFN-, we observed a great batch-to-batch variability in the responses, not reported by previous authors.27,28 Therefore, our results do not exclude the possibility that besides Aß1–40, other APP-derived fragments may play a role in platelet-induced macrophage activation.
In summary, this is the first demonstration of Aß in human atherosclerotic plaques, in which it was found to be present in activated, iNOS-expressing, perivascular macrophages that had phagocytized platelets. The in vitro studies indicate that macrophages become activated after phagocytosis of platelets. They strongly suggest that platelet-derived APP is proteolytically processed to Aß and possibly other peptides, which results in macrophage activation, as indicated by iNOS upregulation. This represents a novel mechanism for macrophage activation in atherosclerotic plaques, which is known to result in matrix degradation as demonstrated by others.3
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Acknowledgments |
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The financial support of the GOA (concerted action University of Antwerp), the Fund for Scientific Research–Flanders (FWO, grants 1.5.206.00 and G.0180.01), the Ministry of the Flemish Community (grant AWI.BIL.98), and the Bekales Foundation is greatly appreciated. Dr Kockx is a recipient of an FWO fund for fundamental clinical research. Dr Jans was sponsored by the Flemish Institute for Improvement of Scientific and Technological Research in Industry (IWT). The authors acknowledge the assistance of Luc Andries (HistoGeneX), Martine De Bie (Levenslijn grant 7.0022.98), Hermine Fret (FWO grant G.0427.02), and Rita Van Den Bossche. Human blood platelet concentrates were kindly provided by the Blood Transfusion Center, University Hospital of Antwerp, Antwerp, Belgium.
Received November 29, 2001; revision received April 10, 2002; accepted April 18, 2002.
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