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J. Biol. Chem., Vol. 277, Issue 33, 29889-29896, August 16, 2002

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Accelerated Phagocytosis of Amyloid- by Mouse and Human Microglia Overexpressing the Macrophage Colony-stimulating Factor Receptor^*

From the Neuroscience Research Laboratories, Department of Psychiatry & Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305-5485

Received for publication, January 28, 2002, and in revised form, April 24, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Microglia surrounding A plaques in Alzheimer's disease and in the APPV717F transgenic mouse model of Alzheimer's disease have enhanced immunoreactivity for the macrophage colony-stimulating factor receptor (M-CSFR), encoded by the proto-oncogene c-fms. Increased expression of M-CSFR on cultured microglia results in proliferation and release of pro-inflammatory cytokines and expression of inducible nitric-oxide synthase. We transfected mouse BV-2 and human SV-A3 microglia to overexpress M-CSFR and examined microglial phagocytosis of fluorescein-conjugated A. Flow cytometry and laser confocal microscopy showed accelerated phagocytosis of A in mouse and human microglia because of M-CSFR overexpression that was time- and concentration-dependent. In contrast, microglial uptake of 1-µm diameter polystyrene microspheres was not enhanced by M-CSFR overexpression. Microglial uptake of A was blocked by cytochalasin D, which inhibits phagocytosis. M-CSFR overexpression increased the mRNA for macrophage scavenger receptor A, and fucoidan blocking of macrophage scavenger receptors inhibited uptake of A. M-CSFR antibody blocking experiments demonstrated that increased A uptake depended on the interaction of the M-CSFR with its ligand. These results suggest that overexpression of M-CSFR in APPV717F mice may prime microglia for phagocytosis of A after immunization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Alzheimer's disease (AD)¹ is characterized by amyloid- peptide (A) plaques surrounded by microglia. A is thought to be directly neurotoxic, and activated microglia are hypothesized to have negative effects on neurons through the release of effectors of inflammation (1). However, as brain macrophages microglia can clear A by phagocytosis, primarily through macrophage scavenger receptors (MSR) (2-6). Immunization of transgenic mice modeling AD with A results in clearance of plaques from the brain (7). Whereas some results suggest that microglial phagocytosis may be key in clearance of A after immunization (8), other findings indicate that circulating antibodies may result in movement of A out of the brain (9). This controversy has stimulated renewed interest in uptake of A by microglia.

A distinctive phenotypic feature of microglia surrounding A plaques in APPV717F transgenic mice and in AD is enhanced expression of the macrophage colony-stimulating factor receptor (M-CSFR), translation product of the c-fms proto-oncogene (10, 11). Microglial M-CSFR expression is also increased after experimental ischemic and traumatic brain injury (12, 13). M-CSFR regulates proliferation, activation, and survival of cells in the monocyte-macrophage lineage through tyrosine kinase activation of diverse signal transduction pathways including: Src kinase, Ras-ERK, phosphoinositide 3-kinase, and p38 MAP kinase (14-16). Deletion of M-CSFR expression results in decreased numbers of cells of the mononuclear phagocyte lineage (17). In AD macrophage colony-stimulating factor (M-CSF), the ligand for M-CSFR expressed by neurons and glia is also increased (18). Simultaneous increases in M-CSF and M-CSFR expression in the brain could result in significant changes in microglial function.

We recently demonstrated that overexpression of M-CSFR by cultured microglia increases proliferation, stimulates release of pro-inflammatory and chemotactic cytokines, and induces a paracrine inflammatory response in a microglial-organotypic co-culture system (19). In the present study we sought to determine the effects of M-CSFR overexpression on A phagocytosis by cultured mouse and human microglia. We hypothesized that M-CSFR-induced activation of microglia would increase their capacity to clear A from culture medium. Although A immunization clinical trials have been discontinued in humans for the present, identifying factors that enhance microglial clearance of A may be of benefit in devising alternative means of decreasing A burden in the brain in AD.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Microglial Cell Lines, Plasmid Transfections, and Tissue culture-- The c-fms expression plasmid pTK1 was a gift from Dr. Rao Tekmal (Emory University, Atlanta, GA), and contains the complete mouse c-fms sequence under the control of an SV40 promoter (20). Transfections were carried out using mouse BV-2 and human SV-A3 microglial cells. The BV-2 immortalized microglial cell line has been characterized previously (21-24). Because of phenotypic changes that occur at higher passages in BV-2 cells,² BV-2 cells used in these experiments were from passage 25 or lower. The human SV-A3 cell line was a gift from Dr. Robert Nelson (Pfizer Central Research, Groton, CT). The SV-A3 line was an immortalized line from fetal brain microglia, and phenotypic features have been previously described (19).

Transient transfections with the pTK1 plasmid were carried out using LipofectAMINE PLUS^TM reagent (Invitrogen). BV-2 or SV-A3 cells were plated at 65% confluency in 6-well tissue culture Eagle's medium. BV-2 cells were grown in Dulbecco's modified Eagle's medium (Applied Scientific, South San Francisco, CA) supplemented with 5% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 4 mM glutamine (Applied Scientific), 0.02 M HEPES (Applied Scientific), 0.1% penicillin/streptomycin (Sigma). SV-A3 cells were cultured with Dulbecco's modified Eagle's/F-12 medium (Invitrogen) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. For each transfection reaction, 0.2 µg of pTK1 plasmid pre-coated with 6 µl of the PLUS reagent and 5 µl of LipofectAMINE were used per 5 × 10⁵ microglial cells. Control transfections were performed in parallel with an equal volume of LipofectAMINE PLUS reagent only (transfection medium; designated by M in the figures). Transfections using the pZeoSV plasmid (Invitrogen) that contains the pTK1 backbone sequence originally used in c-fms subcloning were included as a second control, although we previously showed that pZeoSV does not induce a microglial proinflammatory response (19). Cells were transfected with the pZeoSV plasmid using the same procedure as described for pTK1. After the addition of transfection reagents, cells were returned to the incubator for 24 h, and then used for phagocytosis studies.

Cell Surface M-CSFR-- Microglia that were transfected with the pTK1 plasmid were then compared with nontransfected microglia for M-CSFR expression to determine transfection efficiency. Immunocytochemical visualization of M-CSFR was demonstrated previously (19). To quantify cells overexpressing M-CSFR, 24 h post-transfection BV-2 cells were washed with PBS buffer, reacted for 1 h with 10% normal goat serum (Zymed Laboratories Inc.), and incubated with rabbit anti-mouse M-CSFR antiserum (1:500) (Upstate Biotechnology Inc., Lake Placid, NY) for 16 h at 4 °C. The M-CSFR antibody has specificity for the extracellular domain of the receptor (25). Subsequently cells were incubated for 1 h at 37 °C with a 1:500 dilution of Cy3-labeled goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA). Cells were then washed three times with 1× PBS buffer on ice, then detached with trypsin and collected by centrifugation. Finally, cells were resuspended in 0.5 ml of 1% paraformaldehyde solution and analyzed for M-CSFR overexpression by flow cytometry as described below. A population of 10,000 cells per sample was used to acquire two-dimensional contour forward and orthogonal scatter diagrams. The nonviable subpopulation of cells was excluded by setting the gated forward scatter value to 60. Cellular autofluorescence in the Cy3 channel was 4.32 units for control and c-fms-transfected microglia.

Phagocytosis Assay for Fluorescein-conjugated A-- Microglial phagocytosis of A was determined using Fluo-A, a direct fluorescein conjugate that can be visualized with a flow cytometer or with fluorescence microscopy (PerkinElmer Life Sciences). Immediately before use, Fluo-A 1-40 or 1-42 was resuspended in 2% DMSO solution in Earl's buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.9 mM MgCl₂, 25 mM HEPES, pH 7.4) at 200 nM and allowed to aggregate for 2 h at 37 °C according to the manufacturer's instructions. This preparation was similar to that utilized by Li et al. (3). Fluo-A was used at concentrations between 10 and 100 nM in phagocytosis assays.

Another A preparation, similar to that described by Webster et al. (26), was utilized to study phagocytosis at higher A concentrations. This preparation consisted of preaggregated Fluo-A 1-40 added to nonlabeled A 1-40 (Bachem, Torrance, CA) that had been aggregated by 60 h incubation at 4 °C, followed by 8 h incubation with gentle shaking at 37 °C as previously described (24). The stock solution of this preparation consisted of 100 nM Fluo-A 1-40 in 10 µM aggregated unlabeled 1-40. The stock solution was diluted 10-fold in culture medium prior to the phagocytosis assay.

For the phagocytosis assay, mouse BV-2 cells were plated at 65% density in a 6-well tissue culture dish and grown using the conditions described above. After 24 h of transfection with c-fms expression plasmid, the control plasmid pZeoSV, or treatment with transfection medium alone, the medium was removed and BV-2 cells were washed four times with 1× Dulbecco's PBS (Invitrogen). Immediately before the phagocytosis assay, cells were equilibrated for 1 h at 37 °C in 750 µl/well of Earl's buffer that had been supplemented with 0.2% bovine serum albumin (Sigma) and 0.01% glucose (Mallinckrodt, Paris, KY). Subsequently, Fluo-A reagents were added and cells were incubated at 37 °C in the dark.

To monitor the time course and concentration dependence of Fluo-A phagocytosis in mouse microglia, nontransfected BV-2 cells were treated with Fluo-A at final concentrations of 10, 20, or 100 nM, and harvested after 30 min, 60 min, or 24 h of incubation. To examine the effects of M-CSFR overexpression, BV-2 cells were transfected with the pTK1 plasmid, exposed to 15 nM Fluo-A 1-40, and harvested after 30 min, 60 min, or 24 h. In experiments with SV-A3 human microglia, transfection and phagocytosis assay conditions were the same as those for BV-2 cells, except that the final concentration of Fluo-A was 10 nM and the exposure time was 120 min. To compare phagocytosis of A peptides 1-40 and 1-42, BV-2 microglia transfected with the c-fms plasmid pTK1, the control plasmid pZeoSV, or treated with transfection medium were exposed to 20 nM Fluo-A 1-40 or 1-42 for 120 min. To assess phagocytosis of aggregated A at higher concentrations, preaggregated Fluo-A 1-40 with aggregated unlabeled A 1-40 was applied for 180 min at final concentrations of 10 nM labeled peptide and 1 µM unlabeled peptide in the assay medium. Because rapidly proliferating c-fms-transfected BV-2 cells were not viable for more than few hours in serum-free medium, all 24-h phagocytosis experiments were performed in heat-inactivated serum-containing medium. Control samples for extracellular surface binding of Fluo-A contained nontransfected BV-2 cells that were incubated in triplicate wells with 15 nM Fluo-A for 24 h at 4 °C. To study A uptake by MSR, BV-2 cells were pretreated with 100 µM of the MSR ligand fucoidan (Sigma) for 20 min prior to exposure to 20 nM Fluo-A for 120 min. To demonstrate that uptake of Fluo-A involves phagocytosis, microglia were pretreated with 5 µM cytochalasin D (27) (Sigma) using the same conditions as for fucoidan.

The phagocytosis assay was terminated by placing cells on ice and promptly removing medium containing Fluo-A. Cells were washed 4 times with ice-cold 1× PBS to remove residual unbound Fluo-A, and then detached from the bottom of the wells by treatment for 4 min with 250 µl of trypsin-EDTA (Irvine Scientific, Santa Ana, CA) at room temperature. Trypsinization also served to remove residual amounts of unbound Fluo-A. The cell suspension was transferred to a microcentrifuge tube and centrifuged at 13,000 rpm for 4 min. Finally, pelleted cells were resuspended by gentle pipetting in 500 µl of 1× PBS buffer on ice, transferred to 5-ml polystyrene round-bottom tubes (BD PharMingen), and immediately analyzed by flow cytometry.

Flow Cytometry Analysis-- Flow cytometry was performed using a 10-color flow cytometer, a hybrid instrument consisting of a FACStarPlus bench (BD PharMingen), MoFlo electronics (Cytomation, Fort Collins, CO) and a custom electronics and computer system (Stanford Cell Sorting Facility, Stanford, CA) (28). The emitted fluorescence intensity was measured in triplicate using 10,000 cells per sample. Data were collected on the fluorescence-activated cell sorter desk (36), calibrated so that fluorescence measurements were in the linear range above a value of 1.0. Subsequent statistical analysis of scatter plots and fluorescence histograms was performed using FlowJo 3.3 software (Tree Star Inc., San Carlos, CA). The viability of each sample was evaluated from forward/orthogonal scatter plots obtained on the ungated cell population. In all cases, cell populations were found to be uniform in size and without large aggregates, and the number of nonviable cells and cellular debris was found to be less than 10% of the total. For quantitative analysis, the mean fluorescence values for each sample were collected from the ungated cell population and corrected by subtracting the corresponding cellular autofluorescence value to obtain net signal value. The autofluorescence values for nontransfected and c-fms-transfected microglia were similar and had an average value of 4.27 units, except in Fig. 4A for which the autofluorescence was 5.6. Autofluorescence of human SV-A3 microglia was 3.31 units. Absolute mean fluorescence intensities (given in figure legends) for control microglia were assigned a value of 1.0. Normalized fold change values were then calculated according to the equation: normalized fold fluorescence change = (mean transfected sample fluorescence intensity  cellular autofluorescence)/(mean control sample fluorescence intensity  cellular autofluorescence). Each experiment consisted of cell samples run in triplicate, and each experiment was performed three times. Graphs show mean normalized fold fluorescence change with standard deviations (S.D.) from triplicate experiments relative to transfection medium (M) treated control microglia.

Polystyrene Microsphere Phagocytosis-- To examine microglial uptake of a substrate other than A, we used polystyrene microspheres. The stock suspension of fluorescent 1-µm diameter polystyrene microspheres (blue-green FluoSpheres Fluorescent Microspheres, Molecular Probes, Eugene, OR) contained 1 × 10⁷ microspheres per µl of water with 0.02% thimerosal. BV-2 cells were grown to 65% confluency and transfected with the pTK1 c-fms plasmid as described above. The phagocytosis assay was performed identically to that for Fluo-A except that a suspension containing 0.5 µl of microsphere stock suspension per ml of medium was applied to the BV-2 cells. Fluorescence intensity measurements were taken using flow cytometry after 24 h of incubation, and calculations of fluorescence fold change were performed as described for the Fluo-A assay. As a positive control, Fluo-A experiments were performed in parallel to microsphere uptake experiments using the same batch of cells.

Laser Confocal Microscopy-- To visualize phagocytosis of A by microglia we used laser confocal microscopy. BV-2 and SV-A3 cells were cultured and transfected as described above, except that cells were grown on glass coverslips on the bottom of tissue culture dishes. Cells were treated with 25 nM Fluo-A and incubated for 3 h at 37 °C. Cells were then washed 4 times with 1× PBS buffer at 4 °C and then fixed for 20 min with a 4% paraformaldehyde solution (Sigma) in 1× PBS. The coverslips were mounted on glass slides using SlowFade (Molecular Probes, Eugene, OR), and examined on a Zeiss LSM 510 laser confocal microscope (Carl Zeiss Inc., Thornwood, NY) using a ×63 oil immersion objective. Images were captured in the fluorescein isothiocyanate channel. For comparison of c-fms-transfected and control BV-2 and SV-A3 microglia, images were captured using identical acquisition settings for contrast, brightness, pixel, and pinhole dimensions. Images of microglia without Fluo-A treatment acquired with identical settings were used to assess background autofluorescence.

SYBR Green Real-time Quantitative Reverse Transcriptase-PCR-- Mouse BV-2 microglia were grown to 65% confluency and transfected with the pTK1 plasmid or the pZeoSV control plasmid as described above. After 24 h, total RNA was extracted using the TRIzol reagent (Invitrogen). RNA samples were diluted to 1 µg/µl in RNase-free water, and reverse transcription was performed from 1 µg of RNA as previously described (19). Individual 25-µl quantitative SYBR Green real-time PCR reactions consisted of 5 µl of cDNA (50 ng/µl), 12.5 µl of 2× Universal SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA), and 3.75 µl of 50 nM optimized forward and reverse primers with specificity for the mouse macrophage scavenger receptor type A (MSR-A) mRNA (GenBank^TM accession number AF203781). Primer sequences designed using Primer Express software (PE Applied Biosystems) were: forward⁹³⁸, 5'-GACAAATTGGCTTCCCTGGA-3', and reverse¹⁰⁰¹, 5'-CCCGACCTCCCTGGCTT-3', where numbers indicate the position of the 5' nucleotide. A 64-base pair amplicon was generated using these primers. Quantitative PCR was performed on an ABI 5700 Instrument (PE Applied Biosystems) using a 3-stage program provided by the manufacturer: 2 min at 50 °C, 10 min at 95 °C, and then 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Specificity of the amplification product was confirmed using dissociation reaction plots, with a distinct single peak indicating a single PCR product. Values for each sample were compared using a standard curve (37). Each sample was tested in triplicate PCR wells, and samples obtained from three independent experiments were used to calculate the mean ± S.D.

M-CSFR Antibody Blocking-- BV-2 microglia were grown to a final density of 1 × 10⁵ cells per well in 6-well tissue culture dishes and transfected with the pTK1 c-fms expression plasmid as described above. After 24 h, transfection medium was removed, and after washing cells at 4 °C with 1× PBS buffer, blocking medium was added consisting of 750 µl of serum-free medium and 5 µl of the rabbit anti-mouse M-CSFR antibody (Upstate Biotechnology). Transfected and control microglia were grown in the presence of the M-CSFR blocking reagent for 24 h at 37 °C to account for the greatly increased cellular surface expression of M-CSFR after transfection with pTK1 plasmid (19). As a control, microglial cells were treated with an equal dilution of normal rabbit serum (Sigma). Phagocytosis measurements were taken as described above after 120 min of incubation with 5 nM Fluo-A.

Statistical Analysis-- All experiments were performed with triplicate cell culture samples, and each experiment was replicated on at least three separate occasions. Means of triplicate experiments were compared using two-tailed unpaired t tests, or with one or two-way analyses of variance. For post-hoc comparisons among means, the rigorous Scheffe adjustment for multiple testing was used.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fig. 1 summarizes flow cytometry analysis of M-CSFR overexpression on mouse BV-2 microglia detected using the antibody to the extracellular domain of the receptor. Fig. 1A shows that after 24 h of transient transfection with the c-fms expression plasmid pTK1, there was an increase in Cy3 fluorescence (x axis) from transfected microglia relative to nontransfected cells, demonstrating M-CSFR overexpression. The contour diagrams are based on 10,000 cells per sample, and were gated to exclude nonviable cells with a forward scatter value of 60 and less. Examination of the ungated cell population showed that on average less than 10% of the cells were excluded by gating. The fluorescence histograms shown in Fig. 1B demonstrate that the average Cy3 fluorescence increased from 5.67 in nontransfected cells to 51.5 units in transfected cells. After subtracting the average autofluorescence of 4.32 units, the average net Cy3 intensity increased from 1.35 to 47.18 units. We chose a liberal estimate of 20 for the maximum fluorescence intensity of nontransfected cells. Based on the number of pTK1-transfected microglia that had fluorescence intensities greater than 20, the average transfection efficiency was 89.9%.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Flow cytometry analysis of the M-CSFR overexpression in mouse BV-2 microglia. A, Cy3 emitted fluorescence (x axis) plotted with forward scatter (y axis) of nontransfected (red) and transfected BV-2 microglia overexpressing M-CSFR (blue). The forward gate value was set at 60 to exclude nonviable cells. B, Cy3 histograms of control (red) and c-fms-transfected microglia (blue) demonstrate an increase in mean fluorescence intensity as a result of transfection. Cellular autofluorescence was equal to 4.32. C, overlaid orthogonal and forward scatter diagrams of control BV-2 (red) and c-fms-transfected (blue) microglia.

To compare size, granularity, and overall morphology of transfected and control cells, forward/orthogonal scatter profiles of control and M-CSFR overexpressing BV-2 microglia were overlaid (Fig. 1C). After exclusion of nonviable cells there was complete overlap in the scatter plots. This demonstrates no change in the size or structure of BV-2 microglia because of M-CSFR overexpression.

BV-2 mouse and SV-A3 human microglia readily ingested Fluo-A at low nanomolar concentrations. This response was enhanced by overexpression of M-CSFR. Fig. 2 shows uptake of Fluo-A by nontransfected BV-2 cells, measured after 30 and 60 min and 24 h at concentrations of 10, 20, and 100 nM. Uptake occurred in a time- and Fluo-A concentration-dependent manner; a maximum mean normalized fold fluorescence change of 6.64 ± 0.04 was measured in the 100 nM Fluo-A-treated sample for 24 h. Two-way analysis of variance revealed significant effects because of concentration, time, and a time by concentration interaction (all p < 0.001). Post-hoc Scheffe tests showed that overall the increase after 24 h was significantly different from that after 30 and 60 min (Scheffe adjusted p < 0.05). The 30- and 60-min time points did not differ significantly. Overall, the 10, 20, and 100 nM concentrations gave significantly different results (adjusted p < 0.05). The interaction was because of a significantly greater increase at the 100 nM concentration after 24 h than after 30 and 60 min.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Time- and concentration-dependent phagocytosis of the Fluo-A peptide. Mouse BV-2 microglia were treated with 10, 20, and 100 nM concentrations of Fluo-A 1-40 as described under "Experimental Procedures," and phagocytosis was quantified by flow cytometry after 30 min, 60 min, and 24 h of incubation. Uptake of Fluo-A is presented as mean normalized fold fluorescence increase relative to the fluorescence intensity measured in 30 min for a 10 nM sample. Values represent the mean ± S.D. from three independent experiments. Cellular autofluorescence was 4.27 in this and subsequent figures unless noted otherwise; mean fluorescence intensity assigned to the normalized value of 1 was 4.92 units.

Phagocytosis of A was sharply increased after microglia were transiently transfected with the pTK1 c-fms plasmid. Fig. 3 shows confocal imaging of Fluo-A in control and c-fms-transfected microglia, and demonstrates an increase in intracellular Fluo-A in mouse BV-2 (Fig. 3, A and B) and in human SV-A3 (Fig. 3, C and D) cells as a result of M-CSFR overexpression (Fig. 3, B and D). Uptake of Fluo-A by nontransfected SV-A3 cells was extremely low (Fig. 3C), making it difficult to acquire detailed images of the nontransfected state, even with confocal microscopy.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Increased Fluo-A phagocytosis in microglia overexpressing MCSFR. Laser confocal images of mouse BV-2 (A and B) and human SV-A3 (C and D) microglia treated with transfection medium (A and C) or c-fms transfected (B and D) and exposed to 25 nM Fluo-A for 180 min. Serial optical sectioning was used to select images through the interior of cells containing nuclei to demonstrate internalization of Fluo-A.

Increased phagocytosis of Fluo-A because of M-CSFR overexpression was quantified by flow cytometry. Fig. 4A shows a fluorescence histogram from a representative experiment utilizing 10,000 BV-2 microglia that were c-fms transfected (red) or nontransfected (blue), and exposed to 25 nM Fluo-A for 180 min. pTK1-transfected microglia showed no difference in basal autofluorescence from control cells because of their similar size and shape. A positive shift along the x axis indicates an increase in mean fluorescence intensity from 8.7 units in nontransfected to 15.5 units in transfected microglia, which after subtracting the cellular autofluorescence value of 5.6 units results in a net increase of 3.2-fold for c-fms-transfected microglia. Fig. 4B shows data from triplicate experiments in which BV-2 cells were transfected to overexpress M-CSFR, and fluorescence intensity was measured after 30 min, 60 min, and 24 h of exposure to 15 nM A. Analysis of variance showed significant main effects for transfection, for time, and a transfection by time interaction (all p < 0.001). Overall, c-fms overexpression significantly increased ingestion of A. However, the difference between transfected and nontransfected cells was greater at 24 h than at 60 and 30 min. Flow cytometry measurements taken after 5, 10, and 15 h of incubation showed a progressive increase in Fluo-A ingestion by c-fms-transfected cells, although uptake was not as great as at 24 h (data not shown). Increased phagocytosis of Fluo-A because of M-CSFR overexpression was also observed with flow cytometry using SV-A3 human microglia. As shown in Fig. 4C, an average 13.75 ± 3.57-fold increase in mean normalized fluorescence was observed in SV-A3 after c-fms transfection (p < 0.005).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Flow cytometry analysis showing increased A phagocytosis in microglia overexpressing M-CSFR. A, fluorescence histograms of Fluo-A phagocytosis in transfection medium-treated (blue) and c-fms-transfected (red) BV-2 microglia demonstrate positive shift upon overexpression of M-CSFR. Histograms are from a representative experiment in which cells were treated for 180 min with 25 nM Fluo-A. The x axis represents on a logarithmic scale fluorescence intensity with respect to relative cell number (y axis). B, uptake of 15 nM Fluo-A measured after 30 min, 60 min, and 24 h from triplicate samples of transfected and nontransfected cells. The normalized value of 1 was equal to 4.92 fluorescence units. The right-hand bar shows uptake by BV-2 cells maintained at 4 °C for 24 h during exposure to Fluo-A. C, increased phagocytosis of Fluo-A by human SV-A3 microglia overexpressing M-CSFR. SV-A3 autofluorescence was 3.31 units; the normalized value of 1 was 3.51 units. D, inhibition of Fluo-A phagocytosis in c-fms-transfected microglia after treatment with cytochalasin D. Treated cells showed a fold change of 0.32 in comparison with nontreated cells; the normalized fluorescence value of 1 equals 6.23 units.

To verify that fluorescence was because of internalized rather than surface-bound Fluo-A, BV-2 microglia were incubated for 24 h at 4 °C before flow cytometry. Fig. 4B shows that surface bound Fluo-A showed a normalized fold fluorescence change of 0.45 ± 0.07. Because microglia incubated with Fluo-A for the same amount of time at 37 °C had a 2.73 normalized fold fluorescence change value, cell surface A accounts for only 16.6% of signal at the point of maximal Fluo-A internalization. Furthermore, Fluo-A uptake upon c-fms transfection was dramatically reduced by microglial pretreatment with the phagocytosis inhibitor cytochalasin D as shown in Fig. 4D (p < 0.0001).

Nontransfected and c-fms-transfected microglia avidly ingested a 1 µM concentration of unlabeled aggregated A 1-40 containing a final 10 nM concentration of Fluo-A (Fig. 5A). The uptake of Fluo-A by nontransfected microglia was significantly greater when it was presented in combination with the 1 µM aggregated A than when it was presented alone at a 10 nM concentration (p < 0.002). When cells were transfected to overexpress M-CSFR, there was a further significant increase in uptake of the 1 µM aggregates (p < 0.02). BV-2 cells also readily ingested 1-µm diameter fluorescent polystyrene microspheres. However, there was no significant difference between control cells and cells overexpressing M-CSFR in uptake of the microspheres after 24 h (Fig. 5B). Similar results were obtained after 90-min, 10-h, and 15-h incubations (data not shown). Parallel experiments performed with the same batch of cells showed a large increase in uptake of Fluo-A after c-fms transfection.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Increased phagocytosis of micromolar concentrations Fluo-A. Polystyrene microsphere uptake. Comparison of A 1-40 and A 1-42 uptake. A, increase in mean fluorescence in control and c-fms-transfected BV-2 microglia after 180 min exposure to 10 nM Fluo-A in 1 µM aggregated A 1-40. Results are presented in parallel to cells treated with 10 nM Fluo-A alone. Nontransfected BV-2 cells ingested the 1 µM A preparation more avidly than 10 nM Fluo-A alone. Transfection resulted in a further increase in phagocytosis of both preparations. The normalized fluorescence value of 1 equals 5.34 units. B, uptake of 1-µm diameter fluorescent polystyrene microspheres by BV-2 cells after 24 h of incubation. There was no difference in microsphere uptake between cells treated with transfection medium and c-fms-transfected cells. C, comparison of Fluo-A 1-40 and 1-42 uptake. The increase in uptake after c-fms transfection was similar for both peptides. BV-2 cells transfected with the control plasmid pZeoSV were similar to medium-treated control cells. Normalized fold fluorescence of 1 equals 5.17 units.

M-CSFR overexpression resulted in increased phagocytosis of Fluo-A 1-42 as well as Fluo-A 1-40. Fig. 5C shows results from BV-2 cells treated with transfection medium, c-fms transfected, and transfected with the control vector pZeoSV. Fluo-A 1-42 and Fluo A 1-40 were internalized by microglia with similar efficiency, and c-fms transfection increased ingestion of both peptides (Scheffe adjusted p < 0.05). Transfection with the control vector pZeoSV that contains the complete DNA backbone of the pTK1 plasmid but lacks the c-fms cDNA did not significantly increase phagocytosis of either peptide over values from transfection medium-treated control cells.

To determine whether the M-CSFR-induced increase in Fluo-A phagocytosis was dependent on MSR, BV-2 microglia were treated with the MSR ligand fucoidan prior to the addition of Fluo-A. As shown in Fig. 6A, fucoidan resulted in a significant decrease in mean phagocytosis in c-fms-transfected (p < 0.05) and nontransfected (p < 0.05) cells, although uptake was not abolished. Fig. 6B shows that overexpression of M-CSFR on BV-2 microglia also resulted in a significant increase in MSR-A mRNA as determined with real-time reverse transcriptase-PCR (p < 0.03).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Macrophage scavenger receptor blocking and expression analysis. A, fucoidan treatment resulted in a similar decrease in phagocytosis of Fluo-A in c-fms-transfected (from 2.14 to 0.93-fold) and control BV-2 cells (from 1.00 to 0.49-fold), indicating involvement of MSR under both conditions. The normalized fluorescence value of 1 equals 5.76 units. B, induction of MSR-A mRNA in BV-2 microglia overexpressing M-CSFR. Real-time quantitative reverse transcriptase-PCR results were normalized using glyceraldehyde-3-phosphate dehydrogenase as a callibrator gene and are shown as average expression fold change (with standard deviations) relative to MSR-A mRNA in pZeoSV transfected cells (left-hand bar). Overexpression of M-CSFR in BV-2 microglia resulted in an average 1.92-fold expression increase of MSR-A mRNA in comparison to control cells that were transfected with the control plasmid pZeoSV.

To demonstrate that increased Fluo-A phagocytosis in c-fms-transfected microglia was dependent on interactions of the M-CSFR with its ligand, M-CSFR antibody blocking experiments were performed. As shown in Fig. 7, BV-2 microglia exposed to the M-CSFR blocking antibody showed a decrease in A phagocytosis relative to cells without blocking (Scheffe adjusted p < 0.05), and relative to cells treated with normal rabbit serum (adjusted p < 0.05). There was no significant difference between untreated and serum-treated cells in Fluo-A uptake.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Reduced Fluo-A phagocytosis in c-fms-transfected BV-2 microglia after blocking of M-CSFR. Antibody blocking of M-CSFR resulted in a decrease in Fluo-A uptake to 0.25 of that seen in transfected cells with no blocking. Normal rabbit serum (NRS) had no significant effect. The normalized fluorescence value of 1 equals 7.72 units.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

These results demonstrate that overexpression of the M-CSFR on cultured mouse and human microglia results in increased phagocytosis of A. Recently, there has been great interest in microglial phagocytosis of A because microglia may clear A from the brain in APPV717F mice after immunization (7, 8). Microglia in APPV717F mice show increased M-CSFR expression (10). Increased microglial M-CSFR expression may be a key factor in the ability of APPV717F microglia to efficiently clear A from the brain after immunization.

We used a direct fluorescein conjugate of A to detect microglial phagocytosis by flow cytometry and confocal microscopy. The kinetics of BV-2 murine microglial phagocytosis of Fluo-A were similar to those reported for primary microglia derived from adult human brain (3). We also observed that BV-2 mouse and SV-A3 human microglia rapidly ingest A at concentrations in the range of 10 to 100 nM. These concentrations are similar to those found in APPV717F brain homogenates from animals younger than 8 months of age (29). Experiments using nanomolar A concentrations may model conditions encountered by microglia in APPV717F mice immunized beginning early in life, a treatment that largely prevents A deposition (7). Immunization is also effective in decreasing, although not eliminating, A deposits when initiated in older APPV717F animals. We showed that increased expression of M-CSFR is also effective in enhancing microglial phagocytosis of A at the micromolar concentrations likely present in older transgenic mice. Furthermore, M-CSFR overexpression enhances microglial ingestion of the A 1-40 and 1-42 species, both of which are present in AD and transgenic mice modeling AD.

MSR-A are important in microglial internalization of A (4, 26). Pretreatment of BV-2 microglia with the MSR ligand fucoidan resulted in a decrease in the uptake of Fluo-A by nontransfected cells, and a proportionately similar decrease in uptake by cells overexpressing M-CSFR. This indicates that the increase in phagocytosis of A because of M-CSFR occurs in part through MSR. The accelerated uptake of A after c-fms transfection may have been due in part to the increased MSR-A expression we detected in transfected cells. Others have also reported increased MSR-A expression after activation of the M-CSFR (30). Uptake of A not blocked by fucoidan may have been because of interactions between A and other microglial receptors. Although we used serum-free medium or media containing heat-inactivated serum, microglia express C1q and its receptor (31), which could also have been involved in uptake of A (26). These results suggest that increased M-CSFR expression activates multiple pathways involved in the internalization of A.

Cytochalasin D inhibits actin polymerization and hence interferes with phagocytosis (27). We found that cytochalasin D treatment resulted in an ~70% reduction in A ingestion by BV-2 cells overexpressing M-CSFR. This level of A phagocytosis inhibition is similar to that observed with cytochalasin D for primary rodent microglia (26). These results, along with those showing low cell surface binding and confocal images showing Fluo-A internalization, demonstrate that M-CSFR-induced internalization is phagocytic.

BV-2 cells have been shown to avidly ingest 1-µm fluorescent microspheres by phagocytosis (22, 32). Surprisingly, uptake of microspheres was not enhanced by M-CSFR overexpression. This could mean that overexpression of M-CSFR results in preferential uptake of A aggregates. However, additional phagocytic substrates such as Candida albicans, Escherichia coli, myelin, and zymosan will need to be tested to determine whether M-CSFR overexpression results in A-specific uptake.

Increased A phagocytosis in microglia overexpressing M-CSFR is dependent on interaction with its ligand, M-CSF, as shown by antibody blocking of the extracellular domain of the M-CSFR on BV-2 cells. This is of significance in that M-CSF levels are up-regulated in AD brain (18), and hence could augment increased phagocytosis induced by increased M-CSFR expression. Furthermore, A increases expression of M-CSF by microglia (33) as does M-CSFR overexpression (19). Antibody blocking of the M-CSFR did not completely abolish the increase in phagocytosis induced by c-fms transfection. The very strong increase in M-CSFR induced by c-fms transfection in BV-2 cells (19) most likely resulted in continual recruitment of new receptors to the cell surface, making complete blocking difficult. Furthermore, there is evidence for intracytoplasmic autocrine interactions between M-CSF and the M-CSFR (34). These autocrine interactions, which could also occur in microglia overexpressing c-fms in AD, would not be affected by extracellular antibody blocking experiments.

Microglia have been implicated in clearance of A in transgenic mice after immunization (7, 8). However, a competing hypothesis holds that circulating A antibodies do not enter the brain but rather form a "sink" that results in net transport of A from the central nervous system to the circulation (9). If microglia do play a significant role in A clearance after immunization, anti-A antibody levels in the brain are likely to be very low. Our data suggest that microglia in AD and in APPV717F mice that overexpress M-CSFR are likely to be "primed" to rapidly ingest A. This may explain why small amounts of antibody entering the brain after vaccination can have a major effect on A clearance.

We did not determine the fate of A taken up by microglial cells overexpressing M-CSFR. A taken up by cultured microglia could be degraded, remain intact intracellularly (35), or be processed and released in a form with a modified propensity for aggregation. In APPV717F mice, whatever the fate of A internalized by microglia, the area occupied by extracellular aggregated A decreases after A immunization (7). A immunization clinical trials in humans were recently halted because of a "central nervous system inflammatory state." Although the nature of this reaction is unclear, it is conceivable that in AD patients increased activation of microglia near A deposits after immunization could contribute to an inflammatory meningitis or encephalitis. We are currently examining the effects of A on cultured microglia overexpressing M-CSFR, because these may differ from the effects previously reported for nontransfected microglia (24).

In summary, overexpression of the M-CSFR by microglia resulted in enhanced phagocytosis of A, but not of polystyrene microspheres. M-CSFR-induced phagocytosis of A occurred at a wide range of peptide concentrations, and both A 1-40 and 1-42 were ingested with equal avidity. These effects were dependent on interactions between the M-CSFR and its ligand, and in part on MSR. The role of microglia in clearance of A after immunization of APPV717F mice has been questioned because of the low A antibody titers likely to exist in the brain (9). However, our results suggest that microglia in APPV717F mice that overexpress M-CSFR should show aggressive uptake of A even at low intracerebral antibody titers. Experiments are in progress to determine the effects of M-CSFR overexpression on microglial uptake of opsonized A. If opsonization results in a further enhancement of A uptake by cultured microglia overexpressing M-CSFR, then it is unlikely that high antibody concentrations are required for clearance of A by microglia after immunization of APPV717F mice.

	ACKNOWLEDGEMENTS

We thank Dr. Rajeshwar Rao Tekmal, Emory University School of Medicine, for providing pTK1 c-fms expression plasmid; Dr. Robert Nelson, Pfizer Central Research, Groton, CT, for providing human SV-A3 microglia, and Grace Perez, Clara Poon, and Feifei Zhao for technical assistance. We also thank Mark Gilbert, Dr. David Parks, and the staff of the Stanford Shared Cell Sorting Facility for assistance with flow cytometry and data analysis, and Dr. Robert Malenka and the Nancy Pritzker laboratory for assistance with confocal microscopy.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants MH57833, MH40041, and AG17824 and the Alzheimer's Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Published, JBC Papers in Press, May 24, 2002, DOI 10.1074/jbc.M200868200

To whom correspondence should be addressed: Dept. of Psychiatry & Behavioral Sciences, MSLS P-104, Stanford University School of Medicine, Stanford, CA 94305-5485. Tel.: 650-725-0565; Fax: 650-725-5714; E-mail: gmurphy@stanford.edu.

² O. M. Mitrasinovic and G. M. Murphy, Jr., unpublished observations.

	ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; M-CSFR, macrophage colony-stimulating factor receptor; M-CSF, macrophage colony-stimulating factor; A, amyloid ; Fluo-A, fluorescein-conjugated amyloid ; PBS, phosphate-buffered saline; MSR-A, mouse scavenger receptor type A.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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