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Originally published online as doi:10.1189/jlb.1203620 on May 10, 2004

Published online before print May 10, 2004
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(Journal of Leukocyte Biology. 2004;76:451-461.)
© 2004 by Society for Leukocyte Biology

Uptake of Aß 1–40- and Aß 1–42-coated yeast by microglial cells: a role for LRP

Vincent Laporte*, Yves Lombard*, Rachel Levy-Benezra*, Christine Tranchant*,{dagger}, Philippe Poindron*,1 and Jean-Marie Warter*,{dagger}

* Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, EA 3429, Faculté de Pharmacie, and
{dagger} Clinique Neurologique 2, Service des maladies du Nerf et du Muscle, Hôpitaux Universitaires, Faculté de Médecine, Université Louis Pasteur de Strasbourg, Cedex, France

1Correspondence: Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, EA 3429, Faculté de Pharmacie, Université Louis Pasteur, 74, route du Rhin-BP 24, F-67401 Illkirch, Cedex, France. E-mail: poindron{at}pharma.u-strasbg.fr


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ABSTRACT
 
Artificial diffuse and amyloid core of neuritic plaques [ß-amyloid peptide (Aß) deposits] could be prepared using heat-killed yeast particles opsonized with Aß 1–40 or Aß 1–42 peptides. Interaction and fate of these artificial deposits with microglial cells could be followed using a method of staining that allows discrimination of adherent and internalized, heat-killed yeast particles. Using this system, it was possible to show that nonfibrillar or fibrillar (f)Aß peptides, formed in solution upon heating (aggregates), could not impair the internalization of heat-killed yeast particles opsonized with fAß 1–40 or fAß 1–42. This indicated that depending on their physical state, Aß peptide(s) do not recognize the same receptors and probably do not follow the same internalization pathway. Using competitive ligands of class A scavenger receptors (SR-A) or low-density lipoprotein-related receptor protein (LRP), it has been shown that SR-A were not involved in the recognition of amyloid peptide deposits, whereas LRP specifically recognized deposits of fAß 1–42 (but not fAß 1–40) and mediated their phagocytosis.

Key Words: amyloid ß-protein • senile plaque • Alzheimer’s disease • scavenger receptor A • phagocytosis • endocytosis


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INTRODUCTION
 
Alzheimer’s disease (AD), the most frequent form of dementia in the elderly, is characterized in the brain by several histological features and especially senile plaques [1 ]. There are two major forms of senile plaques: the neuritic plaques, which consist of an extracellular amyloid deposit or a central core composed of fibrillar ß-amyloid peptides (fAß) surrounded by dystrophic neurites, reactive astrocytes, and activated microglia, and the diffuse plaques, which principally contain nonfibrillar Aß peptide (nfAß) and are devoid of amyloid structure. Aß peptides are 40 or 42 amino acid-long fragments (Aß 1–40 and Aß 1–42, respectively), resulting from the cleavage of a membrane-spanning glycoprotein, ß-amyloid precursor protein (APP). Many studies have demonstrated that production, aggregation, and deposition of Aß are key events in AD pathogenesis. It has been shown that diffuse plaques consist almost exclusively of Aß 1–42 and truncated (17–42) APP-derived peptide and that neuritic plaques contain Aß 1–40 and Aß 1–42 [2 , 3 ]. Aß 1–42 is therefore the predominant form. In vitro and in vivo, Aß 1–42 is less soluble and forms amyloid fibrils more easily than Aß 1–40. An increase in Aß production and/or a defect in Aß clearance might be responsible for Aß deposition [4 ].

To prevent neurotoxicity of Aß peptides, whichever their conformation and physical state, an organism can clear them using four mechanisms: transport of nfAß and fAß peptides in excess through the blood-brain barrier [5 ]; clearance of fAß peptides through receptor-mediated endocytosis [6 ]; extracellular catabolism of nfAß and fAß peptides [4 , 7 ], especially through the action of neprilysin, a membrane-anchored, neutral endoprotease [8 ]; and pinocytosis of extracellular nfAß peptides [9 ]. Improper clearance of Aß may play a major role in AD pathogenesis [4 , 10 ]. Microglial cells migrate and surround the Aß deposits [11 ]. The presence of activated (possibly by Aß) microglial cells within senile plaques suggests that these cells may contribute to AD pathogenesis by removing or producing amyloid fibrils. Microglial cells are cerebral, phagocytic mononuclear cells. They are primarily involved in inflammatory response of the brain. Activated microglial cells, indeed, are able to release cytotoxic agents such as cytokines [12 ], proteases [13 ], nitric oxide [14 ], or reactive oxygen intermediates [15 ]. Although the available data suggest that activated microglial cells are mainly harmful in AD, one cannot exclude that they are also involved in the removal and possibly digestion of amyloid fibers. The role of microglial cells in AD pathogenesis would be ambivalent. It has been proved that cultured microglial cells can internalize small aggregates of fAß [6 , 9 , 16 17 18 19 ]; injection of fAß aggregates into the rat striatum lead to their phagocytosis by microglial cells [14 ]; and microglial cells from brains of patients with AD are stained with anti-Aß peptide antibodies [20 21 22 23 ]. Uptake of Aß is mediated by various receptors, such as class A scavenger receptors (SR-A) [17 ], Fc receptors [24 ], and low-density lipoprotein receptor-related protein (LRP), provided that Aß is complexed to {alpha}2-macroglobulin ({alpha}2M) [25 ], lactoferrin [25 ], or apolipoprotein E (APO-E) [26 ], which are ligands of LRP.

To study the interactions of Aß peptides with microglial cells in vitro, we have developed an experimental system consisting of heat-killed yeast particles opsonized with Aß 1–40 or Aß 1–42 peptides (subsequently, shortly referred to as Aß 1–40- and Aß 1–42-opsonized, heat-killed yeast particles, respectively). We have used four different forms of Aß peptides: soluble peptides (aqueous solution); aggregates of fibrillar peptides (heated aqueous solution); deposits of nf peptides (nfAß-opsonized, heat-killed yeast particles); and deposits of f peptides (fAß-opsonized, heat-killed yeast particles). nfAß-opsonized, heat-killed yeast particles mimicked diffuse senile plaques, and fAß-opsonized, heat-killed yeast particles mimicked amyloid core of neuritic senile plaques. We supposed that microglial cells could internalize Aß peptides through receptor-mediated endocytosis or through phagocytosis, depending on the size (aggregates or deposits) of the preparation used. By using competitive ligands, it was possible to explore interactions between fAß aggregates or deposits and several kinds of microglial cell receptors and follow the fate of opsonized, heat-killed yeast particles.

In this work, we present results, which validate the use of Aß-opsonized, heat-killed yeast particles as artificial diffuse or amyloid plaques; indicate that these artificial deposits could be ingested by microglial cells using different receptors; and show that LRP is partly involved in phagocytosis of amyloid deposits.


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MATERIALS AND METHODS
 
Chemicals and reagents
Aß and related peptides (15–25, 25–35, 35–25, 1–40, and 1–42) were obtained from Bachem (Voisins-le-Bretonneux, France). They were dissolved in milli-Q water (1 mg/ml), aliquoted, and stored at –80°C. Laminarin (soluble ß-glucan extracted from Laminaria digitata), {alpha}-mannans (extracted from Saccharomyces cerevisiae), lactoferrin, {alpha}2M, and methylamine were purchased from Sigma (L’Isle d’beau-Chesnes, France). 125I-laleled Aß 1–40 and Aß 1–42 peptides were obtained from Amersham France SA (Les Ulis). Tannic acid was obtained from Merck (Darmstadt, Germany). Receptor-associated protein (RAP) was a kind gift from S. K. Moestrup (Department of Clinical Chemistry, AKH Aarhus University Hospital, Aarhus, Denmark).

Activation of {alpha}2M
To expose its receptor recognition site, {alpha}2M was incubated in aqueous solution consisting of methylamine (200 mmol/l), sodium phosphate (50 mmol/l), sodium chloride (150 mmol/l), pH 7.4, for 1 h at room temperature. Methylamine was removed by extensive dialysis against a 0.1 mmol/l sodium bicarbonate, 0.5 mmol/l sodium chloride, pH 8.3, aqueous solution. The methylamine-treated {alpha}2M will be subsequently referred to as {alpha}2M+.

Electron microscopy
Amyloid fibrils were obtained by incubation of Aß in aqueous solutions at 37°C. Formation of fibrils was checked at regular intervals by negative staining of the heated preparations with a 2% uranyl acetate aqueous solution and observation with a Philipps EM 410 electron microscope.

Congo red staining
fAß preparations were mixed with a 6.6% solution of Congo red (C.I 22120, Sigma) in a mixture (v/v) of phosphate-buffered saline (PBS; 60%) and ethanol (40%) in such a way that the final concentration of Congo red was 6 µM and that of Aß, 90 µg/ml, as described [27 ]. The mixture is made directly in wells of 96-well plates. The plates were maintained for 15 min at room temperature, and the absorbance of the mixture was recorded at 340 nm, 405 nm, 450 nm, 492 nm, 540 nm, 570 nm, 600 nm, and 620 nm.

Yeast
Heat-killed yeast suspension was prepared from a local strain of S. cerevisiae. Briefly, yeast was grown in Sabouraud’s broth for 48 h at 28°C under agitation and then autoclaved for 45 min at 120°C in the culture medium. Heat-killed yeast suspension was washed three times in PBS without calcium and magnesium (PBS–). The stock suspension contained ~4 x 109 particles/ml. It was aliquoted and stored at 4°C until use.

Preparation of Aß-opsonized, heat-killed yeast particles
Heat-killed yeast suspension (200x106 particles) was centrifuged for 6 min at 3200 g. Supernatant was removed, and the pellet of heat-killed yeast particles was mixed with 20 µl fAß (or in some cases, nfAß) peptide aqueous solution. After an overnight incubation at room temperature, the heat-killed yeast suspension was washed three times in PBS and stored in PBS. To study the stability of the Aß peptide binding to the heat-killed yeast particles, aqueous solutions of 125I-labeled nfAß 1–40 or nfAß 1–42 peptides were mixed to cold, aqueous solutions of nfAß 1–40 or nfAß 1–42 peptides in a molar ratio of 1 to 20,000 at a total concentration of 1 mg/ml and allowed to transform into fAß as described above. Heat-killed yeast particles were then opsonized with these radioactive preparations. Heat-killed, yeast-associated radioactivity was measured at regular intervals by {gamma}-counting, after three washes in PBS.

Microglial cells
The microglial cell line used throughout this study was established from a newborn BALB/c mouse cortex freed of meninges, as described for obtaining peritoneal macrophage cell lines [28 ]. After cortices were cut in pieces less than 1 mm3, the explants were transferred into 50 ml centrifugation tube, and the volume was adjusted to 30 ml per brain with RPMI-1640 medium (Life Technologies, Cergy-Pontoise, France) containing 10% inactivated fetal calf serum (FCS; Life Technologies), 105 U/l penicillin, 50 mg/ml streptomycin (Laboratoires Diamant, Puteaux, France). The resuspended explants were transferred into 25 cm2 plastic-culture flasks (Poly Labo Paul Block et Cie, Strasbourg, France) and incubated at 37°C under 5% CO2. Cells were regularly subcultured as described [28 ]. Briefly, cultured cells were mostly adherent. However, some cells spontaneously passed into the culture medium (~50,000 cells/ml culture medium; viability exceeding 98%, as assessed by trypan blue exclusion test). These cells were identified as microglial cells (Fig. 1 ) by their reactivity with the biotin-conjugated Griffonia (Bandeiraea) simplicifolia isolectin B4 (Sigma) [29 ]. Cells passed into suspension were allowed to adhere to 14-mm-diameter, sterile, glass coverslips placed in 24-well plates (Nunclon, Poly Labo Paul Block et Cie). Under these conditions, cells adhered very rapidly to the support. The plates were incubated for 24 h at 37°C in an air (95%)–CO2 (5%)-humidified incubator. Microglial cell preparations were then treated or not with cytochalasin B (Sigma) before phagocytosis. For cytochalasin treatment, they were washed with PBS and incubated for 20 min in PBS containing the appropriate cytochalasin B concentration.



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  		Figure 1. Identification of microglial cells by staining with biotin-conjugated G. simplicifolia isolectin B4. (a) Aspect of microglial cells after incubation with biotin-conjugated lectin and revelation with streptavidin-conjugated peroxidase. (b) Control cells: aspect of a cell not treated with lectin before adjunction of streptavidin-conjugated peroxidase. Note the stellate outlines of these cells.

The immortalized murine BV-2 cell line was grown and maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 105 U/l penicillin, and 50 mg/ml streptomycin. Cells (70–80% confluent) were harvested, seeded into 24-well plates containing glass coverslips, and incubated for 24 h at 37°C.

Phagocytosis
In a previous study, we had observed that heat-killed yeast particles were ingested by murine macrophages in culture through a phagocytic pathway involving mannose and ß-glucan receptors and called lectinophagocytosis [30 ]. To investigate the specific phagocytosis of Aß-opsonized, heat-killed yeast particles by microglial cells, it was therefore necessary to block the normal phagocytic pathway of heat-killed yeast particles by ligands of mannose and ß-glucan receptors, namely soluble {alpha}-mannans and laminarin, respectively.

Just before addition of Aß-opsonized, heat-killed yeast suspension, the cells were washed once with PBS and treated with 450 µl PBS containing laminarin (0.4 mg/ml), {alpha}-mannans (0.4 mg/ml), and the competitor ligand to be studied at the required concentration. Ten minutes later, 30 µl Aß-opsonized, heat-killed yeast (~50 particles per cell) was added to each well. The plates were incubated for 45 min at 37°C in an air (95%)–CO2 (5%)-humidified incubator. The cells were then washed three times with PBS. To discriminate adherent and ingested Aß-opsonized, heat-killed yeast particles, the preparations were stained as described [31 ]. Briefly, the cells were treated for 1 min with a 1% tannic acid solution in PBS, washed with PBS, stained with May-Grünwald Giemsa solutions, and then mounted, cell-side down, on glass slides. Adherent, noningested Aß-opsonized, heat-killed yeast appeared as violet particles and ingested Aß-opsonized, heat-killed yeast, as pink particles (Fig. 2 ). At least 100 microglial cells were observed per coverslip at a magnification of 1,000. Data were expressed in terms of mean number of Aß-opsonized, heat-killed yeast particles ingested (MNI) or adhering (MNA) per cell, that is, the total number of ingested or adhering particles counted divided by the total number of cells observed.



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  		Figure 2. Discrimation between adhering (dark) and ingested (clear), heat-killed yeast particles by the Giaimis method [31 ].

Phagocytosis of radioactive, Aß-opsonized, heat-killed yeast particles
To verify that Aß-opsonized, heat-killed yeast particles remained coated with amyloid fibrils after being ingested by microglial cells, radioactive, Aß-opsonized, heat-killed yeast particles were prepared as described above and added to microglial cells, pretreated for 10 min with {alpha}-mannan and laminarin. After a 45-min incubation, the cells were washed with PBS and mechanically lysed. Cell-associated, heat-killed yeast particles were then collected and washed three times with PBS. Yeast-associated radioactivity was measured by {gamma}-counting.

Internalization of free fAß
To verify that free fAß were readily ingested, microglial cells were incubated for 55 min with radioactive fAß. Unbound amyloid fibrils were collected by washing three times the preparations, cell-surface-bound fibrils were removed by treating the cells for 10 min with a solution of 0.2 mol/l acetic acid containing 0.5 M NaCl, and internalized fibrils were collected by dissolving the cells in boiling 1 mol/l NaOH for 1 h. Then, internalized, cell-surface-bound and unbound Aß were determined by {gamma}-counting.

Opsonization of Aß-opsonized, heat-killed yeast particles by free fAß during competitive experiments
To verify that Aß-opsonized, heat-killed yeast particles do not adsorb the free fAß used as competitor in some experiments, fAß-opsonized, heat-killed yeast particles (5x106 particles) were incubated for 45 min with radioactive amyloid fibril (total concentration of fAß=10–6 mol/l). Particles were washed three times with PBS to remove fibrils. Supernatant radioactivity and yeast-associated radioactivity were evaluated by {gamma}-counting.


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RESULTS
 
Soluble Aß peptides form fibrils upon heating
In a preliminary set of experiments, we determined the conditions that allow Aß and related peptides to form amyloid fibrils, which represent their pathological conformation. It was shown that the Aß 25–35 peptide formed fibrils following incubation at 37°C for 1 day, whereas it took 5 days for Aß 1–40 and Aß 1–42 to adopt this conformation. Neither Aß 15–25 nor Aß 35–25 was able to form fibrils under the same experimental conditions. Figure 3 shows the appearence of fibrils formed by the different peptides used. It should be underlined that our fAß peptide solutions were never turbid, indicating that fAß aggregates obtained in this way were of small size and never reached that of a senile plaque, whereas aggregates obtained by heating under agitation can reach a mean diameter of ~15 µm [14 ].



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  		Figure 3. Appearance of fibrils formed upon heating of soluble Aß peptides for 5 days at 37°C. (a–c) Unheated solutions. (e–g) Heated solutions. (a, e) Aß 25–35. (b, f) Aß 1–40. (c, g) Aß 1–42. (d, h) Heated solutions of Aß 15–25 or Aß 35–25, respectively. Note that fibrils (diameter: 0.01–0.02 µm) formed only with Aß 25–35, Aß 1–40, or Aß 1–42 peptides. (i) Absorption spectra of Congo red solution ({square}) or Congo red in the presence of fAß (Ab) 1–40 (•) or fAß (Ab) 1–42 ({circ}). Note the characteristic, spectral shift of Congo red, which proves the amyloid structure of the heated fibrillar Aß peptides.

Congo red binds to amyloid proteins because of their ß-sheet conformation. This binding is accompanied by a modification in the absorption spectrum. As shown in Figure 3 , after incubation, Aß 1–40 and Aß 1–42 transformed into amyloid fAß structures, as they induced the specific spectral shift expected.

fAß peptides bind to heat-killed yeast particles
To determine whether Aß peptides were able to bind to heat-killed yeast particles, mixtures of cold and radio-iodinated nfAß or fAß aqueous solutions were added to heat-killed yeast particles initially washed with PBS-. The mixtures were kept at room temperature overnight. The suspensions were then centrifuged at 3,200 g for 6 min, and the pellets were washed three times in PBS to eliminate unbound Aß. Radioactivity of resulting suspensions was assayed on an aliquot, and the results were referred to as zero-time radioactivity. The same operations were done on the remaining suspension every day after zero time. Figure. 4a and 4b , shows that fAß 1–40, fAß 1–42, and nfAß 1–42 could bind to heat-killed yeast particles, whereas nfAß 1–40 could not (zero time). However, binding of nfAß 1–42 is rather low. After 3 days, ~50% of fAß 1–40 and fAß 1–42 remained bound. Therefore, it was possible to use fAß 1–40- and fAß 1–42-opsonized, heat-killed yeast particles as artificial amyloid plaques and nfAß 1–42-opsonized, heat-killed yeast particles as artificial diffuse plaques.



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  		Figure 4. Heat-killed yeast particles can be opsonized by Aß peptides. (a) A mixture of 125I-labeled nfAß (nfAb) 1–40 and cold nfAß 1–40 was allowed to transform into amyloid fibrils (•) or not (fAb; {square}). Heat-killed yeast particles were then treated by these radioactive preparations. Yeast-associated radioactivity was measured at regular intervals by {gamma}-counting after three washes in PBS. (b) Same experiment using 125I-labeled nfAß 1–42 and cold nfAß 1–42.

Ingestion of fAß-opsonized, heat-killed yeast particles by microglial cells
Under our experimental conditions, that is, after having inhibited lectin-like, receptor-mediated phagocytosis, the following results were obtained with Aß-opsonized, heat-killed yeast particles (Fig. 5 ). Aß 1–42-opsonized and Aß-treated, heat-killed yeast particles were readily ingested whether the Aß peptides were heated or not before opsonization or treatment. It is worthy to note that fAß 25–35-treated or fAß 1–42-opsonized, heat-killed yeast particles were ingested at a higher rate than nfAß 25–35-treated or nfAß 1–42-opsonized, heat-killed yeast particles, indicating that fully fibrillar forms of these peptides were more easily internalized than their nonfibrillar or uncompletely fibrillar forms (Aß 25–35 and to a lesser extent, Aß 1–42 have a tendency to spontaneously form some fibrils in solution); moreover, fAß 1–40-opsonized, heat-killed yeast particles were heavily ingested, whereas when treated with nfAß 1–40, heat-killed yeast particles were ingested at the same rate as untreated, heat-killed yeast particles. Laminarin and {alpha}-mannans not only almost completely inhibited lectin-like, receptor-mediated phagocytosis of heat-killed yeast particles but ingestion of Aß 35–25- or Aß 15–25-treated, heat-killed yeast particles, whether these peptides were heated or not before the treatment. From these results, we concluded that when heat-killed yeast particles were opsonized with fAß 1–40 or fAß 1–42, they can be ingested by a process involving specific receptors.



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  		Figure 5. Ingestion of Aß-opsonized, heat-killed yeast particles or heat-killed yeast particles treated with different Aß-related peptides (Ab). After a 24-h culture on glass coverslips, microglial cells were pretreated with inhibitors of a lectin-like, receptor-mediated ingestion pathway and were then incubated for 45 min with heat-killed yeast particles treated with different Aß-related peptides preincubated (solid bars) or not (open bars) at 37°C. Ingested particles were counted on 100 cells randomly selected, and three wells were observed for each condition. Data are expressed as the MNI ± SEM. ***, P < 0.001; **, P < 0.01.

However, an increase in phagocytosis of Aß-opsonized, heat-killed yeast particles didn’t prove that Aß-opsonized, heat-killed yeast particles were ingested by microglial cells through Aß-specific receptors in the presence of {alpha}-mannan and laminarin. To ensure that Aß-opsonized, heat-killed yeast particles were ingested in their opsonized state, experiments of phagocytosis were performed with radioactive, fAß-opsonized, heat-killed yeast particles in the presence of {alpha}-mannan and laminarin, and radioactivity was measured. As shown in Figure 6 , a significant amount of cell-associated radioactivity could be detected, indicating that Aß-opsonized, heat-killed yeast particles were adhering or ingested by microglial cells.



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  		Figure 6. After a 24-h culture on glass coverslips, microglial cells were pretreated for 10 min with inhibitors of lectinophagocytosis and were then incubated for 45 min with [125I]fAß-opsonized (Ab), heat-killed yeast particles. Microglial cell-associated particles were collected and washed, and radioactivity of suspension was measured by {gamma}-counting. Error bars represent SEM. **, P < 0.01; *, P < 0.05.

Figure 7 shows that cytochalasin B, a drug known to depolymerize actin and inhibit phagocytosis, prevented ingestion of Aß 1–40-opsonized, Aß 1–42-opsonized, and Aß 25–35-treated, heat-killed yeast particles. These results confirmed that Aß-opsonized, heat-killed yeast particles enter microglial cells through a phagocytotic process.



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  		Figure 7. Effect of cytochalasin B on ingestion of fAß-opsonized, heat-killed yeast particles by microglial cells. After a 24-h culture on glass coverslips, microglial cells were pretreated for 30 min with variable concentrations of cytochalasin B, then treated with lectinophagocytosis inhibitors for 10 min, and incubated for 45 min with fAß 1–40-opsonized, heat-killed yeast particles (a) or fAß 1–42-opsonized, heat-killed yeast particles (b). Ingested and adhering particles were counted on 100 cells randomly selected, and three wells were observed for each condition. Data are expressed as the MNI and the MNA ± SEM. Note that cytochalasin B diminished the number of ingested particles but did not modify the number of adhering particles. ***, P < 0.001; *, P < 0.05. Statistics were performed for each value versus untreated cells.

Ingestion of fAß-opsonized, heat-killed yeast particles by microglial cells is not inhibited by nfAß and fAß peptides
Under our experimental conditions, phagocytosis of Aß 1–40- or Aß 1–42-opsonized, heat-killed yeast particles was not inhibited in the presence of soluble or fibrillar forms of Aß 1–40 or Aß 1–42 (Fig. 8 ), supporting the hypothesis that Aß peptides can enter microglial cells by using different ways that depend on their physical and/or conformational states.



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  		Figure 8. Free fAß and nFAß peptides do not apparently inhibit ingestion of fAß-opsonized, heat-killed yeast particles by microglial cells. After a 24-h culture on glass coverslips, microglial cells were pretreated for 10 min with inhibitors of lectinophagocytosis and fAß peptide (•) or nf Aß peptide ({square}), then incubated for 45 min with fAß 1–40 (Ab 1–40)-opsonized, heat-killed yeast particles (a) or fAß 1–42 (Ab 1–42)-opsonized, heat-killed yeast particles (b). Competitors used are Aß 1–40 (a) and Aß 1–42 (b). Ingested particles were counted on 100 cells randomly selected, and three wells were observed for each condition. Untreated cells serve as a control and represent 100% of phagocytosis. Error bars represent SEM. Statistics were performed for each value versus untreated cells, and no significant difference was found. (E–n: E minus n)

To confirm this hypothesis, we verified that microglial cells were able to ingest free fAß, and free fAß did not bind to Aß-opsonized, heat-killed yeast particles during the experiments of competition. As shown in Figure 9 , radioactive, free fAß was partly ingested by microglial cells, the major part of free peptide remaining extracellular. It was therefore possible that absence of competition between free fAb and Aß-opsonized, heat-killed yeast particles be attributed to adsorption of free extracellular fAß onto Aß-opsonized, heat-killed yeast particles. To exclude this possibility, fAß-opsonized, heat-killed yeast particles were incubated in solution of radioactive fAß for 45 min (duration of the phagocytosis test), and the radioactivity associated to fAß-opsonized, heat-killed yeast particles or remaining in solution was measured (Fig. 10 ). Radioactivity associated to fAß-opsonized, heat-killed yeast particles was shown to be negligible, which proved that under these conditions, free fAß cannot bind to Aß-opsonized, heat-killed yeast particles.



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  		Figure 9. Ingestion of free amyloid fibrils by microglial cells. After a 24-h culture on glass coverslips, microglial cells were incubated for 55 min with radioactive fAß 1–40 (a) or fAß 1–42 (b). Ingested ({Delta}), cell-surface bound ({blacksquare}), or extracellular ({diamondsuit}) fAß was collected and measured by {gamma}-counting. Error bars represent SEM.



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  		Figure 10. Adsorption of free fAß on fAß-opsonized, heat-killed yeast particles during competition experiment. fAß-opsonized, heat-killed yeast particles were incubated for 45 min with free, radioactive fibrils of Aß. Then, fAß-opsonized, heat-killed yeast particles and radiolabeled fAß were separated by centrifugation, and radioactivity of the pellet of fAß-opsonized, heat-killed yeast particles (open bars) or supernatant (solid bars) was measured by {gamma}-counting. The experiment was done with fAß 1–40-opsonized, heat-killed yeast particles and fAß 1–40 (Ab 1–40) or fAß 1–42-opsonized, heat-killed yeast particles and fAß 1–42 (Ab 1–42). Note that fAß-opsonized, heat-killed yeast particles do not absorb free fAß. Error bars represent SEM. ***, P < 0.001.

Role of SR-A in ingestion of fAß-opsonized, heat-killed yeast particles
SR-A are involved in the clearance of many different denatured ligands [32 ] and in the elimination of Aß from culture medium [17 ]. This is why we initially searched whether they were responsible for ingestion of fAß-opsonized, heat-killed yeast particles. Fucoidin or dextran sulfate is known to be a ligand of SR-A [32 ]. We therefore studied the effects of these compounds on ingestion of fAß-opsonized, heat-killed yeast particles by microglial cells in the presence of laminarin and {alpha}-mannans. Figure 11 shows that none of these SR-A ligands was able to inhibit ingestion of fAß 1–40- or fAß 1–42-opsonized, heat-killed yeast particles.



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  		Figure 11. Ligands of SR-A do not inhibit phagocytosis of fAß-opsonized, heat-killed yeast particles by microglial cells. After a 24-h culture on glass coverslips, microglial cells were pretreated for 10 min with lectinophagocytosis inhibitors and fucoidin (a) or dextran sulfate (b) and were then incubated for 45 min with fAß 1–40 (Ab 1–40)-opsonized, heat-killed yeast particles (•) or fAß 1–42 (Ab 1–42)-opsonized, heat-killed yeast particles ({circ}). Ingested particles were counted on 100 cells randomly selected, and three wells were observed for each condition. Untreated wells serve as a control and represent 100% of phagocytosis. Error bars represent SEM. Statistics were performed for each value versus untreated cells, and no significant difference was found.

Role of LRP in ingestion of fAß-opsonized, heat-killed yeast particles
As mentioned in the introduction, LRP can recognize some secreted forms of ß-amyloid precursor protein and mediates their internalization. This phenomenon can lead to degradation of these peptides. We therefore attempted to determine whether LRP was involved in ingestion of fAß-opsonized, heat-killed yeast particles. For this purpose, lectin-like receptors of microglial cells were blocked by laminarin and {alpha}-mannans, and ligands known to recognize LRP, namely lactoferrin, {alpha}2M+, or the 39-kD antagonist RAP, were added to the cultures before addition of fAß 1–40- or fAß 1–42-opsonized, heat-killed yeast particles. Figure 12a shows that in the presence of lactoferrin, ingestion of fAß 1–40-opsonized, heat-killed yeast particles was not impaired, whichever the concentration of the competitor ligand used, whereas that of fAß 1–42-opsonized was inhibited to a degree depending on that concentration. Similar observations (Fig. 12b) were made when {alpha}2M+ was used. At a concentration of 300 nmol/l, ingestion of fAß 1–42-opsonized, heat-killed yeast particles was inhibited by ~50%, whereas that of fAß 1–40-opsonized, heat-killed yeast particles remained unaffected. In addition, RAP (at a concentration of 100 nmol/l) also partly inhibited (20%) phagocytosis of fAß 1–42-opsonized, heat-killed yeast particles but did not reduce that of fAß 1–40-opsonized, heat-killed yeast particles (Fig. 12c) .



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  		Figure 12. Differential effects of ligands of LRP on phagocytosis of fAß-opsonized, heat-killed yeast particles by microglial cells. After a 24-h culture on glass coverslips, microglial cells were pretreated for 10 min with lectinophagocytosis inhibitors and lactoferrin (a), {alpha}2M+ (b), or RAP (c) and were then incubated for 45 min with fAß 1–40 (Ab 1–40)-opsonized, heat-killed yeast particles (•) or fAß 1–42 (Ab 1–42)-opsonized, heat-killed yeast particles ({circ}). Ingested particles were counted on 100 cells randomly selected, and three wells were observed for each experiment. Untreated wells serve as a control and represent 100% of phagocytosis. Error bars represent SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05. Statistics were performed for each value versus untreated cells.

Similar results were obtained using the BV-2 microglial cell line. Figure 13 shows that lactoferrin or {alpha}2M+ effectively competes with fAß 1–42-opsonized, heat-killed yeast particles for LRP from BV-2, as elimination of LRP ligands before introduction of fAß 1–42-opsonized, heat-killed yeast particles does not modify the amount of particles ingested by BV-2.



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  		Figure 13. Ingestion of fAß 1–42-opsonized, heat-killed yeast particles by BV-2 microglial cell line. After a 24-h culture on glass coverslips, BV-2 were pretreated with inhibitors of lectin-like, receptor-mediated ingestion pathway and ligands of LRP, were then washed three times with PBS+ (open bars) to remove unbound inhibitors, and were incubated for 45 min at 37°C with fAß 1–42-opsonized, heat-killed yeast particles and lectinophagocytosis inhibitors. Controls were done as follow: BV-2 were pretreated with {alpha}-mannans, laminarin, and LRP ligand and were then incubated for 45 min with fAß 1–42-opsonized, heat-killed yeast particles (solid bars). Error bars represent SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05.


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DISCUSSION
 
The present study shows that it is possible to use Aß-opsonized, heat-killed yeast particles for studying phagocytosis of Aß peptide deposits by microglial cells. This conclusion is supported by the following facts. Nf- and fAß 1–42 and fAß 1–40 could bind to heat-killed yeast particles. Ingestion of fAß 1–40- and fAß 1–42-opsonized, heat-killed yeast particles was blocked by cytochalasin B, a substance known to inhibit phagocytosis [33 ].

fAß-opsonized, heat-killed yeast particles could be structured as amyloid deposits. These deposits consist of thin Aß fibers, perpendicularly bound to long fibers of heparan sulfate proteoglycan, the external part of which is enriched in electronegative, superficial charges of heparan sulfate [34 ]. The negative charges probably facilitate binding of Aß fibers. Here, we show that fAß 1–40 and fAß 1–42 peptides were able to bind to heat-killed yeast particles deprived in calcium and magnesium by washing with PBS–.

The conformational and physical states of Aß peptides are very likely the main features that will determine their fate (pathway of internalization and intracellular processing). We show here that aggregates of Aß peptides do not use the same internalization pathway as deposits to enter microglial cells: Indeed, neither free fAß 1–40 nor fAß 1–42 peptides could compete for microglial Aß peptide receptors with fAß 1–40- or fAß 1–42-opsonized, heat-killed yeast particles. Therefore, it is probable that the receptors involved in internalization of aggregates (fAß) or deposits (fAß-opsonized, heat-killed yeast particles) of fAß peptides are not identical; fAß 1–40 and fAß 1–42 deposits are phagocytosed by microglial cells through different receptors. The molecular mechanisms of receptor-mediated endocytosis and phagocytosis are quite different and depend on the size of elements to be internalized [35 ]. The mode of ingestion probably will determine the intracellular fate of Aß peptides, as these peptides can be directed to different populations of endosomes, depending on their mode of internalization [36 ]. That Aß peptides use the receptor-mediated endocytic pathway is proved by numerous studies. In vitro, Aß peptides can be cleared from culture medium by several cell types, such as smooth muscle cells [37 ] or neuroblastoma cells [38 ], which are unable to phagocytose particulate substances. Monocytes [39 ] and microglial cells [9 , 16 , 40 ] were also proved to clear Aß peptides from culture medium. However, in these studies, the size of aggregates was not always mentioned, so that it is difficult to draw definite conclusions on the mechanism of ingestion. Whatever the way of entry, it seems, however, that Aß peptides can persist several days in cells. Ingested fAß peptides remained 20 days in phagosomes [41 ] and 12 days in endosomes [9 ].

Our results show that nf and fAß 1–42- and fAß 1–40-opsonized, heat-killed yeast particles could be internalized by microglial cells through a phagocytic mechanism (effect of cytochalasin B) involving receptors differing from those used by nonopsonized, heat-killed yeast particles (that is, mannose and ß-glucan receptors). We attempted, therefore, to identify the receptors involved in ingestion of fAß-opsonized, heat-killed yeast particles. To date, indeed, the receptors possibly involved in elimination of fibrillar, potentially neurotoxic, Aß peptides are not known. SR-A have been suspected to play a role in Aß peptide clearance: In vitro, indeed, SR-A of microglial cells have been shown to bind aggregated Aß 1–42 peptides [17 ]. However, other studies did not support this hypothesis. It has been observed, for instance, that fAß peptides did not bind to SR-A of human peripheral blood monocytes in vitro [42 ]. It has also been found that deletion of SR-A in a murine model of AD reduced neither the amount of amyloid deposit nor neurodegeneration [43 ]. A number of other receptors are suspected to play a role in Aß clearance, such as formyl peptide receptor (FPR) or receptor for advanced glycation end-products (RAGE). The role of these receptors is rather indirect, as binding of Aß to FPR or RAGE induces a release of chemokines [44 , 45 ] that recruit phagocytic microglia and improve the uptake of Aß [46 , 47 ]. We observe here that fAß 1–40- and fAß 1–42-opsonized, heat-killed yeast particles were not recognized by these receptors, as they did not compete for them with fucoidin or dextran sulfate. It is possible (although unlikely) that recognition of aggregated Aß 1–40 or Aß 1–42 peptides observed in vitro resulted from the poor specificity of SR-A, which recognize a broad range of ligands, provided that they are denaturated. SR-A indeed are multiligand receptors that bind various proteins and lipoproteins modified by glycation, acetylation, or oxidation [32 ].

Recent work has shown that LRP is involved in the clearance of soluble Aß 1–40 and/or Aß 1–42 peptides, provided that they (it) are (is) complexed to one of their (its) ligands, namely APO-E [26 ], lactoferrin, or {alpha}2M [25 ]. LRP is a multifunctional cell-surface receptor expressed by microglial cells [48 ]. It could play a role in AD pathogenesis by participating in uptake and degradation of ß-amyloid precursor protein [49 ] and modulating the severity and age of onset of AD in relation to the polymorphism of exons 3 and 6 [50 , 51 ]. Moreover, two of its ligands, APO-E [52 ] and {alpha}2M [53 , 54 ], have been identified as genetic risk factors for late-onset AD; both ligands binds Aß in vitro and mediate its internalization, and several ligands of LRP can be detected in senile plaques, using immunostaining, especially APO-E, {alpha}2M, lactoferrin, lipoprotein lipase, tissue, and urokinase-like plasminogen activators and plasminogen activator inhibitor-1 [55 , 56 ]. Finally, it has been shown that a low expression of LRP in brain is associated with AD [57 ]. In these studies, it is postulated that nfAß and fAß are internalized through receptor-mediated endocytosis or possibly fluid-phase endocytosis. It has also been shown that decreased levels of LRP in brain correlated with increased formation of senile plaques, suggesting that LRP modulate Aß peptide deposition and is involved in its clearance [57 ]. However, it has never been suggested that LRP was implicated in the direct clearance of fAß 1–40 or 1–42 amyloid deposits. fAß 1–42-opsonized, heat-killed yeast particles could partly compete with lactoferrin, {alpha}2M+, or RAP for LRP. This indicated that LRP probably can directly bind amyloid deposits of fAß 1–42 peptide, provided it is correctly presented. However, competitor ligands never succeeded in totally inhibiting ingestion of fAß 1–42-opsonized, heat-killed yeast particles. There should therefore exist another receptor responsible for their residual ingestion. By contrast fAß 1–40-opsonized, heat-killed yeast particles did not compete with lactoferrin, {alpha}2M+, or RAP. It is therefore very probable that fAß 1–40 cannot recognize LRP. Whether the receptors involved in this phenomenon are the same as those responsible for the residual ingestion of fAß 1–42-opsonized, heat-killed yeast particles in the presence of lactoferrin, {alpha}2M+, or RAP remains to be determined. It is the first observation showing that fibrillar Aß 1–40 and Aß 1–42 deposits can be processed by microglial cells through different phagocytic pathways. These results confirm the role of LRP in the Aß metabolism.


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ACKNOWLEDGEMENTS
 
We gratefully acknowledge Dr. S. K. Moestrup for his generous gift of purified RAP.

Received December 8, 2003; revised March 30, 2004; accepted March 31, 2004.


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