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Originally published online as doi:10.1189/jlb.0705377 on December 19, 2005

Published online before print December 19, 2005
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(Journal of Leukocyte Biology. 2006;79:596-610.)
© 2006 by Society for Leukocyte Biology

Gene expression changes by amyloid ß peptide-stimulated human postmortem brain microglia identify activation of multiple inflammatory processes

Douglas G. Walker*,1, John Link{dagger}, Lih-Fen Lue*, Jessica E. Dalsing-Hernandez* and Barry E. Boyes{dagger}

* Laboratory of Neuroinflammation, Sun Health Research Institute, Sun City, Arizona; and
{dagger} Agilent Technologies, Wilmington, Delaware

1 Correspondence: Sun Health Research Institute, 10515 West Santa Fe Drive, Sun City, AZ 85351. E-mail: douglas.walker{at}sunhealth.org


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ABSTRACT
 
A central feature of the inflammatory pathology in Alzheimer’s disease is activated microglia clustered around aggregated amyloid ß (Aß) peptide-containing plaques. In vitro-cultured microglia can be activated to an inflammatory state by aggregated Aß with the induction of a range of different neurotoxic factors and provide a model system for studying microglia Aß interactions. Gene expression responses of human postmortem brain-derived microglia to aggregated Aß were measured using whole genome microarrays to address the hypothesis that Aß interactions with human microglia primarily induce proinflammatory genes and not activation of genes involved in Aß phagocytosis and removal. The results demonstrated that Aß activation of microglia induced a large alteration in gene transcription including activation of many proinflammatory cytokines and chemokines, most notably, interleukin (IL)-1ß, IL-8, and matrix metalloproteinases (MMP), including MMP1, MMP3, MMP9, MMP10, and MMP12. All of these genes could amplify ongoing inflammation, resulting in further neuronal loss. Changes in expression of receptors associated with Aß phagocytosis did not match the changes in proinflammatory gene expression. Time-course gene expression profiling, along with real-time polymerase chain reaction validation of expression changes, demonstrated an acute phase of gene induction for many proinflammatory genes but also chronic activation for many other potentially toxic products. These chronically activated genes included indoleamine 2,3-dioxygenase and kynureninase, which are involved in formation of the neurotoxin quinolinic acid, and S100A8, a potential proinflammatory chemokine. These studies show that activation of microglia by Aß induces multiple genes that could be involved in inflammatory responses contributing to neurodegenerative processes.

Key Words: calgranulin • cytokine • metalloproteinase


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INTRODUCTION
 
Alzheimer’s disease (AD) is characterized neuropathologically by an age-dependent formation of amyloid ß (Aß)-containing plaques, accumulation of neurofibrillary tangles, and differential neuronal loss in selective brain regions [1 ]. Increased numbers of activated microglia and astrocytes are also a pathological feature of the disease [2 ]. It has been noted repeatedly that microglia, a distinct population of brain-resident macrophages, are most activated in regions showing AD pathology, particularly in association with the aggregated types of Aß plaques, and around neurofibrillary tangles [3 ]. Microglial activation is indicative of ongoing chronic inflammatory processes. From pathological studies about postmortem brain tissue and also from cell culture studies about microglia, it was hypothesized that the production of inflammatory molecules, many of which are directly toxic to neurons, could be playing a role in accelerating the disease [3 ]. Microglial-derived factors were shown to be toxic to neurons, including nitric oxide [4 ], reactive oxygen species [5 ], tumor necrosis factor-{alpha} (TNF-{alpha}) [6 ], a protease-resistant toxin induced by interaction with microglial heparan sulfate [7 , 8 ], complement proteins [9 ], and cathepsin B [10 ].

Support for the inflammatory hypothesis came from retrospective studies about patients with a long-term history of taking nonsteroidal, anti-inflammatory drugs, which showed a lower incidence of AD in these populations [11 ]. However, recent clinical trials of a range of anti-inflammatory agents have generally shown no significant effects [12 , 13 ]. In addition, studies involving immunization of transgenic mice with Aß peptide have shown that stimulating microglia could have therapeutic potential by increasing the removal and degradation of Aß [14 ]. Although microglia can phagocytose Aß in vitro, there is limited evidence from studies about human AD brains of microglia phagocytosis of amyloid plaques in vivo [15 ]. Recent data indicate that proinflammatory activation processes could repress phagocytosis of Aß or complement-opsonized Aß and therefore, prevent degradation and removal of plaque Aß [16 ], and this treatment did not interfere with uptake of antibody-opsonized Aß through the immunoglobulin [Ig; Fc receptor (FcR)] receptors [16 ].

A recent study using a functional genomics approach with Aß-stimulated murine microglial cells identified cathepsin B as a key protein mediating microglial neurotoxicity [17 ]. In the present study, we report on gene expression changes in human microglia isolated from postmortem brain tissues, which were stimulated with aggregated/oligomeric Aß (1-42) and subjected to gene expression profiling using microarray hybridization analysis on oligonucleotide DNA arrays with content covering essentially all expressed human genes. These analyses were carried out using microglia isolated from human brain tissue from three AD cases and two nondemented (ND), normal, elderly cases. With this approach, combined with validation of gene expression changes by real-time polymerase chain reaction (PCR) over different time-points, acute and chronic features of gene expression changes associated with human microglial activation by Aß were revealed.


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MATERIALS AND METHODS
 
Human brain tissues
The Sun Health Research Institute Brain Donation Program (Sun City, AZ) provided human brain tissue samples for microglial isolation. Brain tissue was donated with informed consent procedures approved by the Sun Health Corporation Institutional Review Board. Each brain was examined and diagnosed by a board-certified neuropathologist using established criteria for the diagnosis of AD. Tissue for microglial cultures used in the microarray hybridization analyses were from three AD donors and two ND donors (mean age 72 years and mean postmortem delay of 2.4 h). In addition, microglia were prepared from brain tissues from six additional donors (three AD and three ND; mean age 83 years and mean postmortem delay of 3.1 h), and cDNA prepared from these cells were used in the reported real-time PCR validation studies.

Cell culture
Human microglia were isolated from pieces of superior frontal cortex according to our previously published procedures [18 , 19 ]. Purified microglia were grown in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Herndon, VA) supplemented with 10% low endotoxin fetal bovine serum (Hyclone, Logan, UT) at 37°C under an atmosphere of 95% air, 5% CO2, and 100% humidity for 12–14 days. Three days prior to experimentation, microglia were subcultured into tissue-culture plates (12-well plates) at 1 million microglia/well and once adherent, were incubated in serum-free DMEM for 2 days prior to stimulation so as to bring the cells to a nonactivated phenotype. This procedure is followed, as factors present in serum maintain microglia in a spontaneously activated state and interfere with responses induced by Aß peptide alone.

Aß peptide preparation and microglia stimulation
Aß (1-42; rPeptide, Athens, GA) was used in all experiments. Oligomeric aggregates of Aß used for microglial stimulation were prepared according to published procedures [20 , 21 ]. Lyophilized Aß was first dissolved in 5 mM NaOH, to which was added 1/10 vol 10x phosphate-buffered saline (PBS) to a final concentration of 500 µM peptide. This was incubated for 18 h under neutral pH and physiological ionic strength to form aggregates with microglial-activating properties [20 ]. Aß produced by this treatment showed Thioflavin S reactivity, indicative of the presence of fibrils, but atomic force microscopy demonstrated that oligomeric forms were the most abundant species. Stocks of aggregated Aß were used immediately or stored in liquid nitrogen. No differences in chemical or biological properties were evident for peptide stored in this manner. Each batch of Aß peptide was tested for the presence of endotoxin contamination using a Pyroquant Plus Limulus amoebocyte lysate kit with 0.06 EU/ml sensitivity (Cambrex Bioscience, Walkersville, MD) and contained <0.06 EU/10 µg peptide. Aß (2 µM final concentration, unless otherwise noted) was added to microglial cultures in 1 ml serum-free DMEM for 24 h. Peptide vehicle was added to control wells in parallel. In one series of experiments, microglia were incubated with Aß for 24 h, 48 h, and 72 h.

RNA extraction and labeling
RNA was prepared from vehicle and Aß-treated microglia using the Agilent Total RNA mini kit (Agilent Technologies, Wilmington, DE). Integrity of RNA samples was verified using an Agilent bioanalyzer with the RNA 6000 Nano LabChip kit, and concentration and purity were measured by spectrophotometry. Each RNA sample was converted to cyanine 5 (Cy5)- and Cy3-labeled complementary RNA (cRNA) using the Agilent low RNA input fluorescent linear amplification kit. Samples were purified using the Agilent cRNA cleanup kit, and the concentration of synthesized cRNAs and amount of incorporated dyes were measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE).

Microarray experiments
The first two of the microglial cases were analyzed on the Agilent human IA array, which is composed of 60-mer oligonucleotides representing 20,173 transcripts, and also on the human IB array, which is composed of 60-mer oligonucleotides representing over 19,000 transcripts and expressed sequence tags. Subsequent microarray analyses were carried out with Agilent whole human genome microarrays. This array has 44,000 60-mer oligonucleotide features representing over 41,000 human genes and transcripts. For hybridization experiments, 0.5 µg Cy5- and Cy3-labeled cRNA samples were mixed together, fragmented, and hybridized to the arrays for 17 h according to the manufacturer’s instructions using the components of the Agilent In situ Hybridization Kit Plus. Each sample pair was hybridized competitively to at least duplicate arrays with dye-swap labeling of Cy5 and Cy3. After the hybridization period, the arrays were washed using conditions recommended by the manufacturer and then scanned using an Agilent microarray scanner (Model G2565BA). The fluorescent intensities of each feature were extracted and normalized, and statistical analysis was generated using Agilent feature extraction software. Genes were classified as showing a significant change in expression if a Pvalue threshold (<0.05) met statistical criteria. This was normally met for up-regulated or down-regulated changes of greater than 1.8-fold. The normalized fluorescent data were then imported into Rosetta Resolver gene expression data analysis software (Rosetta Biosoftware, Seattle, WA) for clustering analysis and generation of gene expression plots. Data from five separate human microglia cases were combined for data analysis.

Real-time reverse transcriptase-PCR gene expression analysis
To confirm changes in gene expression levels, real-time PCR analyses of selected transcripts were carried out using cDNAs derived from six additional microglial isolates. For these studies, microglia were stimulated with Aß (1-42) at 0.5 µM and 2 µM for 24 h, 48 h, or for some cases, 96 h and also with interferon-{gamma} (IFN-{gamma}; Peprotech, Princeton, NJ; 100 ng/ml) for certain cases. RNA was extracted from treated microglia as described above and reverse-transcribed with random primers following our published protocols [18 ]. These cDNA samples were used as templates for analysis of the gene expression levels by fluorescent analysis using Applied Biosystems (Foster City, CA) predesigned Taqman gene expression assays, using an Mx3000P (Stratagene, La Jolla, CA) or Prism 7000 (Applied Biosystems) real-time PCR machine. The relative amounts of transcripts were calculated using the standard curve method. Standards were prepared from pooled cDNAs, which were serially diluted at a 1:10 ratio over a 4-log concentration range. Expression levels for the studied genes were normalized using the 18S rRNA levels in each sample.

Detection of expressed proteins
Standard enzyme-linked immunosorbent assay (ELISA) methodology was used to measure amounts of interleukin (IL)-6 and IL-8 secreted by microglia in response to Aß [22 ]. IL-6 and IL-8 capture antibodies, biotinylated detection antibodies, protein standards, and detection reagents for ELISA were obtained from R&D Systems (Minneapolis, MN). Standard Western immunoblot methodology with peroxidase substrate chemiluminescence detection was used to demonstrate indoleamine 2,3-dioxygenase (IDO) in extracts of control and treated microglia [22 ] using a mouse monoclonal antibody to human IDO (Upstate Biotechnology, Lake Placid, NY), followed by an antibody to ß-actin (Sigma-Aldrich, St. Louis, MO) for sample normalization.

Measurement of kynurenine
Concentrations of kynurenine in culture media were measured using Ehrlich’s reagent (2% dimethylamino benzaldehyde in glacial acetic acid) [23 ]. Samples were extracted in one-third volume of 30% trichloroacetic acid, spun at 10,000 g for 10 min, transferred in duplicate to microtiter plates, and then mixed with an equal volume of Ehrlich’s reagent. Absorbance of each sample was measured at 492 nm using culture media as blank and compared with a standard curve using serial dilutions of purified kynurenine, prepared and measured under the same conditions.

Statistical analysis
For validation experiments by real-time PCR, the effects of different treatments were tested by one-way ANOVA, using GraphPad Prism 4 software (San Diego, CA) with Newman-Keuls post hoc test of significance. Individual comparisons were made by t-test where appropriate. All data are expressed as mean ± SEM unless stated otherwise. Significance in treatments is assumed if P < 0.05 is obtained in the appropriate test.


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RESULTS
 
Aß peptide activation of human brain microglia
Upon addition of aggregated Aß peptide to cultures of microglia isolated from AD and ND brains, the cells developed a typical reactive morphology [Fig. 1 , phase contrast microscopy showing representative example of (A) vehicle-treated and (B) Aß (2 µM)-treated microglia]. This reactive morphology can persist for a number of days and is not observed if cells are treated with freshly solubilized Aß at the same concentration nor with the equivalent volume of peptide vehicle solution. At the dose used, neither aggregated nor freshly solubilized Aß treatment results in noticeable microglia cell death. For analysis of differential gene expression for microarray hybridization analysis, total cellular RNA was extracted from these cells at 24 h. In one additional series of microarray analyses, RNA was extracted from paired cultures treated with Aß or vehicle for 24 h, 48 h, and 72 h, respectively. In validation experiments, RNA was prepared from microglia after 24 h, 48 h, and 96 h of Aß or vehicle treatment. Even after 96 h of treatment with Aß, there was no evidence of microglia cell toxicity.


Figure 1
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  		Figure 1. Representative phase contrast photomicrographs showing change in microglial morphology after incubation with Aß (2 µM). Microglia were cultured for 14 days and media exchanged for serum-free media 48 h prior to experiment. Cells were treated with Aß-containing solution (2 µM) or an equal volume of peptide vehicle solution (PBS/NaOH). (A) Morphology of microglia treated with peptide vehicle solution. (B) Morphology of microglia treated with Aß peptide for 24 h. Arrows highlight microglia demonstrating ameboid and vacuole formation responses. Original magnification, 425x.

Features of gene expression responses in human microglia
The whole genome oligonucleotide microarrays for hybridization analysis yielded consistent results with RNA derived from Aß-stimulated and control microglia cultured from five different human donors. In an initial experiment, these same genes were analyzed across two separate microarrays (Agilent human IA and human IB), yielding essentially the same results as later experiments. A scatter plot of the gene expression signatures of microglia treated with Aß is shown in Figure 2 . This combined plot shows the normalized expression ratios of three competitively hybridized and dye-swapped whole genome microarrays. These analyses showed that ~9% of total expressed genes were up-regulated significantly, and ~7.5% were down-regulated significantly. The specificity of the response to Aß can be seen in the fact that most genes did not show significant responses.


Figure 2
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  		Figure 2. Scatter plot demonstrating the proportion of genes up-regulated (red) compared with those down-regulated (green) in cultured microglia following 24 h treatment with 2 µM Aß (1-42). Data extracted from three sets of gene arrays were combined and analyzed using Rosetta Resolver software. The y-axes represents log plot of ratio of change in expression following Aß treatment; x-axes represent log of fluorescent intensity of genes (a measure of gene abundance). Each data point represents mean value of expression changes of triplicate cases with each sample being analyzed with dye-swap arrays (n=6). Blue represents the expression of genes whose expression was not affected significantly by Aß treatment. Selected genes with error bars represent those whose expression was increased or decreased significantly, greater than threefold.

Genes differentially expressed in Aß-stimulated microglia
By the criterion of an arbitrarily chosen greater than threefold change in expression, Aß stimulation induced the expression of 413 ± 137 (mean±SD) genes. By the same criterion, Aß stimulation of microglia resulted in down-regulated expression of a mean of 245 ± 144 genes. A gene list was created using the criterion of including genes coding for known proteins, which are up-regulated (Table 1 ) or down-regulated (Table 2 ) by a mean of greater than threefold in all five microglia cases. The tables present the genes as segregated by known or putative function of the gene products, based on various public and private database designations and literature references. In addition, by this criterion, there were 48 genes coding for proteins of unknown function, which were up-regulated greater than threefold across all cases, and 26 unknown genes, which were down-regulated greater than threefold (data not shown). Comparing the magnitude of responses to Aß between the ND brain-derived microglia (n=2) and AD brain-derived microglia (n=3) by t-test for all of the genes in Tables 1 and 2 demonstrated significantly greater gene expression changes in response to Aß treatment in the AD cases compared with those from the ND cases for adrenomedulin (P=0.039), bone morphogenetic protein-6 (P=0.036), CXCL1 (P=0.032), cathepsin L (P=0.048), IL-1 receptor antagonist (P=0.043), and vitamin D receptor (P=0.017). Expression of all of these genes was up-regulated by Aß stimulation (Table 1) . There were no significant differences in responses for down-regulated genes between microglia from AD cases compared with those from the ND cases (Table 2) .


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  		Table 1. Compilation of Human Microglia Genes Induced More Than Threefold after 24 h of Aß Exposure


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  		Table 2. Compilation of Human Microglia Genes Repressed More Than Threefold after 24 h of Aß Exposure

Validation of microarray results
To confirm microarray results, real-time PCR analyses of gene expression were carried out on genes of interest using cDNA derived from additional human microglia, which had been treated with Aß or the proinflammatory cytokine IFN-{gamma} (Figs. 3 and 4 ). In all cases, real-time PCR analyses confirmed the directional changes in transcript levels observed by microarray hybridization analysis. It can be seen that the lower doses of Aß resulted in more moderate levels of gene expression changes (induction and repression). It was of interest that although the relative expression of many acutely induced genes declined at 48 h compared with 24 h, MMP3 and MMP10 induction increased during this period. Validation of gene expression changes measured by microarray hybridization was also observed for selected genes of interest at a single time-point (IL-1ß, TNFAIP6, ICAM-1, and CD9: Fig. 4 ). Larger changes in expression were generally detected by real-time PCR than by microarray analysis. For example, in response to Aß (2 µM), IL-1ß induction by microarray hybridization analysis was 80.3-fold and 339-fold by real-time PCR (Fig. 4) . Similarly, IDO induction at 24 h by microarray analysis was 44.4-fold and 278-fold by real-time PCR (see Fig. 8 ). These differences are likely a result of the competitive nature of two-color detection microarray hybridization analysis, which will show data compression over the wide dynamic range of gene expression changes in the current experimental system. Significant changes in ICAM-1 expression were also validated, although the magnitude of changes by expression profiling was only 3.4-fold.


Figure 3
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  		Figure 3. Real-time PCR verification of microarray results for selected genes with different doses of Aß and times of treatment. cDNA was prepared from RNA isolated from duplicate samples for each treatment and time-point from two to three different cases. Gene expression was measured by real-time PCR using predesigned Taqman gene expression assays (Applied Biosystems). Expression levels for each sample were normalized for expression of 18S rRNA detected by real-time PCR. Graphs showing fold changes in gene expression of MMP1, MMP3, MMP10, IL-8, and SDF-1 by human microglia stimulated with Aß (0.5 µM or 2 µM) or IFN-{gamma} (100 units/ml) for 24 h and 48 h as compared with control (Con). Results presented represented mean ± SEM. Graphs of expression changes lacking statistical annotation did not achieve P < 0.05 as a result of sample variability but showed a pattern of noticeable expression changes.


Figure 4
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  		Figure 4. Real-time PCR verification of microarray results for selected genes after 24 h treatment. cDNA was prepared from RNA isolated from duplicate samples for each treatment and time-point from three different cases. Gene expression was measured by real-time PCR using predesigned Taqman gene expression assays (Applied Biosystems). Expression levels for each sample were normalized for expression of 18S rRNA detected by real-time PCR. Graphs show fold changes in gene expression of IL-1ß, TNFAIP6, ICAM-1, and CD9 by human microglia stimulated with Aß (0.5 µM or 2 µM) or IFN-{gamma} (100 units/ml) for 24 h. The data showing reduction of expression of CD9 did not reach statistical significance. NS, Not significant.


Figure 8
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  		Figure 8. (A and B) ELISA analysis showing a dose-response effect of Aß on levels of IL-8 (A) and IL-6 (B) secreted by microglia into media. Results represent mean ± SEM pg/ml/105 cells from three separate microglia cases. In comparison with gene array (62-fold induction) and real-time PCR (24-fold induction), secreted levels of IL-8 were induced by 9.6-fold by 2 µM Aß. Although mRNA for IL-6 is detectable in unstimulated cultures, constitutive secretion of protein was not detectable. (C) Western blot gels showing induction of intracellular IDO by treatment of microglia with Aß or IFN-{gamma} for 24 h (IDO, upper gel). Relative levels of ß-actin remained constant in each sample (Actin, lower gel). Gels are representative of three independent experiments. Lane 1, Unstimulated microglia; lane 2, microglia stimulated with 0.5 µM Aß; lane 3, microglia stimulated with 2 µM Aß; lane 4, microglia stimulated with IFN-{gamma} (100 units/ml). (D) Effect of Aß and IFN-{gamma} treatment of microglia cultures for 24 h on concentrations of kynurenine, an intermediate in the metabolism of tryptophan to quinolinic acid, in culture media.

Time course of gene expression changes
To identify genes whose expression by microglia are altered after more than 24 h stimulation (chronically activated), changes in gene expression over the 24-, 48-, and 72-h time-period were analyzed by microarray hybridization using matched samples of cultured microglia derived from the same donor. The pattern of gene expression changes over this time-period generally showed a decreasing effect of Aß (Fig. 5 ), although the induction of many genes remained relatively high. This could be a result of the persistence of Aß within these cells. For example, IL-1ß expression changed from 75.9-fold at 24 h to 46.3-fold induction at 72 h, and IL-8 changed from 86.5-fold to 35.7-fold induction (Fig. 5A) . In general, ILs (Fig. 5A) and MMP (Fig. 5B) showed a monotonic decreasing expression over time, whereas other gene families showed a more complex pattern. Changes in expression of members of the CCL chemokine family (Fig. 5C) showed a different pattern, having a more chronic elevation in expression. With the exception of CCL20, whose large induction showed a similar decline over the 72-h period as the ILs, the other key chemokines illustrated in Figure 5C (CCL2, CCL4, CCL5, CCL7) maintained a relatively constant level of elevated induction of mRNA expression over the 72-h period. Selected TNF-associated genes demonstrated a complex pattern with initially decreasing levels at 48 h and indication of elevation at 72 h (Fig. 5D) . Examination of all induced genes in this series of samples identified five inflammatory-associated genes whose induction showed a progressive increase with time. The genes were S100A8 (also known as calgranulin A or myeloid-related protein 8), CCL17 (also known as thymus- and activation-regulated chemokine), TFPI2, TNFSF7, and S100B (Fig. 5E) . Real-time PCR validation demonstrated that S100A8 mRNA induction by Aß (2 µM) increased from a nonsignificant 1.5-fold at 24 h to 24.6-fold at 96 h (P<0.001; Fig. 6 ). Examination of the time course of gene expression changes for key genes involved in Aß phagocytosis did not show patterns of gene expression change with time, consistent with Aß activation promoting increased Aß phagocytosis (Fig. 5F) . Expression of mRNA for Aß receptors MSR-1, CD36, and CD47 mRNA was down-regulated or not significantly changed over the time-period studied, and CD14 mRNA expression showed a progressive increase with time. Fc{gamma}R1A (FCGR1A) and Fc{gamma}R2A mRNA, which are involved in the uptake of antibody-opsonized Aß, showed different patterns of gene expression changes (Fig. 5F) .


Figure 5
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  		Figure 5. Changes in expression ratios with time of selected groups of genes of interest. Microarray results for selected genes of paired samples from the same human microglia case. Microglia were treated with Aß (2 µM) for 24, 48, and 72 h. RNA was extracted from each set of samples, which were then processed for gene array analysis. Each sample was analyzed in duplicate on separate arrays with dye-swap. (A) Progressive decline with time of acute response cytokines. (B) Comparison of expression patterns of selected MMP genes. (C) Comparison of expression patterns of CCL chemokine family genes. (D) Comparison of expression patterns of TNF-{alpha}-related genes. (E) Expression pattern of selected genes showing progressive up-regulation of expression over time. (F) Expression pattern of selected Aß receptor genes associated with phagocytosis and uptake. MSR1, Macrophage scavenger receptor-1; ITGAM, integrin {alpha}M; NEP, neprilysin.


Figure 6
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  		Figure 6. Real-time PCR results demonstrating change in expression of S100A8 mRNA at 24 h and 96 h. Results confirm the microarray analysis that S100A8 expression increases progressively with time. Results presented represented mean ± SEM.

Pathway analysis of gene expression
Whole genome expression analyses allow for identification of coordinate changes in genes related by function, for example, those involved in a biosynthetic pathway. The most significant pathway for microglial production of a neurotoxic factor is the kynurenine pathway for tryptophan metabolism, alterations of which can lead to increased production of the neurotoxic factor quinolinic acid (Fig. 7 A ). Up-regulated expression of IDO and KYNU without a coordinated increase in KATII would favor the production of increased amounts of toxic quinolinic acid rather than the neuroprotective N-methyl-D-aspartate receptor antagonist kynurenic acid (Fig. 7A) . Gene expression analysis showed a marked increase in expression of IDO mRNA and a significant increase in KYNU mRNA without significant changes in expression of mRNA for KMO, KATII, or HAAO (Fig. 7A) . The chronic nature of the up-regulation of IDO and KYNU mRNA was confirmed by real-time PCR analyses of separate samples. After 96 h of Aß stimulation, IDO mRNA and KYNU mRNA induction was 61.8-fold and 2.6-fold, respectively. Although HAAO was down-regulated by 41.6% at 96 h, the gene expression changes in the pathway confirm the potential for quinolinic acid production.


Figure 7
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  		Figure 7. (A) Fold changes in gene expression of enzymes of the kynurenine pathway in Aß-stimulated microglia. Values obtained from a gene expression profile result from separate microglia cases. Results presented show mean ± SD. Changes in expression of less than twofold up or down were not of a statistically significant difference from unstimulated control levels. (B) Real-time PCR analyses to demonstrate chronic elevation of IDO and KYNU mRNA expression by human microglia. Results demonstrate expression of IDO, KYNU, and HAAO, key enzymes involved in quinolinic acid formation, after 24 h (acute) and 96 h (chronic) exposure to Aß. Data demonstrated that expression of IDO and KYNU mRNA by microglia remained significantly elevated 96 h after stimulation with Aß at doses of 0.5 µM and 2 µM. Results presented represented mean ± SEM.

Analysis of expressed proteins
To determine to what extent protein expression correlated with mRNA expression, the effect of Aß on secretion of IL-8 and IL-6 was measured for microglia from three cases that had been stimulated with 0.5 µM and 2 µM Aß (Fig. 8A and B ). There was a 9.6-fold induction of IL-8 secretion compared with a 62-fold change detected by gene array analyses (Fig. 8A) . IL-6 secretion was also induced significantly by Aß, although a dose-response effect was not observed (Fig. 8B) . Similarly, it was demonstrated that Aß (0.5 and 2 µM) and IFN-{gamma} induced IDO protein in microglia (Fig. 8C) . The consequences of increased IDO expression were demonstrated by increased levels of kynurenine in culture media of Aß- or IFN-{gamma}-stimulated cultures (Fig. 8D) .


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DISCUSSION
 
In vitro activation of cultured microglia by aggregated Aß peptide has been used in a number of studies as a model experimental system to study the interaction of microglia with Aß in AD brains (reviewed in ref. [24 ]). We have been able to model this feature using microglia derived from postmortem human brains, perhaps the most suitable cell type for investigating in vivo inflammatory mechanisms of the elderly human brain. The aim of this present study was to measure global transcriptional changes using this model system with the goal of identifying novel features that might be relevant to AD inflammatory changes. As inflammation in AD is likely to be a chronic feature, the hypothesis of this study was that genes of significance to AD inflammatory changes might be those showing increased or extended responses after chronic exposure to Aß. An additional goal of this study was to examine if the gene expression profiles under these conditions would identify changes in receptors associated with Aß phagocytosis. Although microglia are phagocytic cells, there is limited evidence of microglial phagocytosis of aggregated plaques in AD brains [25 ]. Interaction of inflammation-causing cells with relatively insoluble agents provides a mechanism for establishing chronic inflammation as a result of the persistence of the stimuli. Chronic production of macrophage/microglia products has the potential of causing damage to tissue [26 , 27 ].

Examination of the up-regulated and down-regulated gene lists (Tables 1 and 2) supports the hypothesis that microglia interaction with Aß promotes inflammation as a result of induction of many potentially neurotoxic proinflammatory cytokines and proteases. Expression of mRNAs of proinflammatory cytokines considered significant in AD pathology, such as IL-1{alpha}, IL-1ß, IL-6, IL-8, and CCL2 [monocyte chemotactic protein-1 (MCP-1)], was highly induced by Aß, in agreement with previous reports (reviewed in ref. [3 ]). Large changes in expression of mRNA for more than 10 proinflammatory chemokines were noted. Expression of mRNAs for the chemokines CCL2 (MCP-1), CCL4 (macrophage-inflammatory protein-1ß), CCL5 (RANTES), and CCL7 (MCP-3) remained significantly elevated even at 72 h (Fig. 5C) . The chemokine CCL2 has been demonstrated [28 , 29 ] to coordinate many pathological processes including weakening of the blood-brain barrier and to act as a chemoattractant for circulating leukocytes to enter the brain. Similarly, expression of mRNAs for six MMPs was up-regulated significantly in Aß-stimulated microglia. Analysis by real-time PCR of MMP1, MMP3, and MMP10 mRNAs showed chronic elevation of expression at 48 h, which was confirmed in a set of matched samples from an additional case, which was analyzed by microarray hybridization analysis at 24 h, 48 h, and 72 h (Fig. 5B) . Increased expression of MMP1, MMP3, and MMP9 has been shown in AD brains [30 31 32 33 ]. Increased activity of these enzymes has significant potential to contribute to tissue damage [34 ].

Increased expression of IDO mRNA could be significant, as its gene product is the initial rate-limiting enzyme in the kynurenine pathway, the main route for L-tryptophan catabolism. Activation of this pathway leads to production of a number of biologically active molecules including the neurotoxin and immune modulator quinolinic acid [35 36 37 ]. Increased production of quinolinic acid by Aß (1-42)- but not Aß (1-40)-stimulated human macrophages and fetal microglia was demonstrated recently, although this previous study used doses of Aß 12.5- to 25-fold higher than being reported here [36 ]. Our data showed that IDO and KYNU mRNA expression remained significantly elevated 96 h after stimulation, suggesting these gene products could be potential therapeutic targets. There is now extensive data showing quinolinic acid is increased significantly in human immunodeficiency virus-associated neurological damage, wherein activated microglia are a prominent feature [38 39 40 41 ]. A role for quinolinic acid in AD inflammatory pathology has also been suggested from in vitro studies [41 ]. Other identified novel genes whose mRNA expression was increased significantly in a chronic manner were S100A8, S100B, CCL17, TNFSF7, and TFPI2. S100A8 and S100B are putative ligands of the receptor for advanced glycation end-products [42 ], a proinflammatory-inducing receptor that has been implicated in many chronic inflammatory diseases, including AD [43 44 45 46 ]. It is expected that these gene products might participate in chronic inflammation, such as that ongoing in AD, an established feature of S100A8 and related molecules [47 ].

From our data, there was no clear link between the role of proinflammatory activation and increased expression of mRNAs for receptors and enzymes involved in Aß phagocytosis. A number of different receptors have been implicated to be involved in Aß phagocytosis by microglia including MSR-1, CD36, CD14, {alpha}6ß1 integrin, CD47, and E-series prostanoid receptor 2 (EP2) [48 49 50 51 52 ]. It was recently demonstrated that treatment of microglia with certain proinflammatory cytokines including IL-1ß, TNF-{alpha}, CCL2 (MCP-1), and lipopolysaccharide (LPS) reduced phagocytic uptake of Aß, and their treatment with anti-inflammatory cytokines (IL-4, IL-10) and anti-inflammatory agents, including ibuprofen and an EP2 receptor antagonist, prevented the inhibition of phagocytosis caused by IL-1ß or LPS [16 , 50 ]. Our data have shown that Aß activation of human microglia inhibited expression of MSR-1, CD36, and Fc{gamma}R1A mRNA or did not stimulate mRNA expression of the other potential Aß receptors CD14 or CD47 to a significant extent. A number of enzymes associated with Aß degradation such as neprilysin, insulin-degrading enzyme, plasminogen, and endothelin-converting enzyme-1 [53 54 55 56 57 58 ], which the gene array data demonstrated are expressed by microglia, were also not altered significantly by Aß stimulation using this described model of AD inflammatory pathology.

Although it was not an aim of this study to determine if isolated microglia from AD brains had a different phenotype than those isolated from ND brains, our analyses showed that there were significantly greater increases in gene expression by the AD brain-derived microglia in response to Aß stimulation for a small number of genes. These genes were IL1RN, cathepsin L, vitamin D receptor, chemokine CXCL1, BMP6, and adrenomedullin. The role of these genes in AD inflammatory pathology is unclear, but increased expression of IL1RN could be a significant control mechanism to reduce the consequences of elevated IL-1 production by activated microglia in various conditions with neuronal injury [59 60 61 ] and is also believed to have a significant role in AD pathology [62 ]. Activation of the vitamin D receptor has anti-inflammatory effects on inflammatory cells [63 ], and BMP6 has characterized neurotrophic properties [64 ]. Increased expression of cathepsin L has been observed in activated microglia in AD brains [65 ] and increased expression of CXCL1 in activated microglia in multiple sclerosis brains [66 ]. Future studies about these genes will require the collection and characterization of mRNA and media from microglia isolated from a larger series of AD and ND human brain cases. Constitutive differences between AD and ND microglia have been observed for the levels of secretion of macrophage-CSF and complement C1q [44 , 67 ] and for intracellular calcium responses in response to stimuli with ATP and platelet-activating factor [68 ].

The current study also addressed an aspect of microglial response not widely considered, namely the function and identity of genes down-regulated in a significant manner in response to Aß stimulation (Table 2) . Although this list was smaller than the list of up-regulated genes, it included SDF1 (CXCL12), AIF1, and CD9. SDF1, a ligand for the chemokine receptor CXC chemokine receptor 4, has chemoattractant properties for microglia. In brain tissue, SDF1 has been localized to microglia, although its immunoreactivity in astrocytes and neurons appeared more prominent [69 ]. Increased astrocyte expression of SDF1 was a feature in human immunodeficiency virus-associated dementia complex [70 , 71 ]. Quinolinic acid can increase SDF1 expression by human astrocytes [72 ]. Down-regulation of AIF1 was contrary to expectation, as increased microglial expression of AIF1 has been associated with activated microglia in conditions with brain pathology [73 74 75 ]. Down-regulation of CD9, also known as motility-related protein-1, in response to inflammatory stimulation potentially increases the chemotactic responses of cells such as microglia [76 ].

Candidate genes identified in this study were noticeably different from those in a similar study using BV2 rodent microglia stimulated with soluble Aß [10 ], where increased expression of cathepsin B and cathepsin L, tissue inhibitor of MMP2, cytochrome c oxidase, and AIF1 was a prominent feature. However, in our study, with human postmortem brain microglia as the target cell, only cathepsin L was present among genes up-regulated more than threefold. Changes in cathepsin B mRNA, an enzyme reported to mediate microglial neuronal toxicity, were less than twofold by microarray hybridization analysis in any of the cases used in this present study. This was confirmed by real-time PCR using separate microglial-derived cDNA samples (data not shown).

Whole genome expression-profiling of Aß-stimulated microglia has confirmed previous studies about proinflammatory changes and identified a number of new targets. Our data, using microarray analyses combined with validation using real-time PCR, have indicated that IL-1ß, IL-6, and IL-8 mRNA induction changes appear more associated with an acute-phase response, and a number of mRNAs for CCL chemokines show elevated induction with prolonged chronic Aß exposure, as did MMP1, MMP3, MMP9, and MMP10 mRNA. Elevated expression of mRNAs for IDO and certain other kynurenine pathway enzymes shows the potential involvement of this pathway in AD pathogenesis. The most pronounced chronic Aß induction of expression was for S100A8 mRNA. This gene did not feature on the collection of 24 h-induced genes but by 72 h, showed 14-fold induction by gene array and after 96 h, showed 24.6-fold induction by real-time PCR. As chronic secretion of S100A8 could lead to chronic activation of microglia, further studies about this protein are warranted, as increased microglia expression has been demonstrated in certain neurological conditions, although its role in AD has not been studied extensively [77 78 79 80 ].

Results from these analyses indicate the use of gene-expression profiling for studying the global changes in transcription of sets of genes in this defined cellular model of AD inflammatory pathology. Further studies could include comparing the changes in expression profiles of Aß-stimulated microglia treated with anti-inflammatory agents and also comparing the expression profiles of microglia derived from human AD and ND brains.


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ACKNOWLEDGEMENTS
 
This work was support by a grant from the National Institutes of Health (NIH; AG18345, D. G. W.) and grants from the Alzheimer’s Association (D. G. W., L-F. L.). The operation of the Sun Health Brain Donation Program was supported by NIH Grant P30 (AG19610-01 "Arizona Alzheimer’s Disease Core Center"). We thank Dr. Thomas Beach and Ms. Lucia Sue for providing brain tissues from the Sun Health Brain Donation Program for isolation of human microglia and neuropathological diagnoses of donated brain tissues.

Received July 10, 2005; revised October 31, 2005; accepted November 3, 2005.


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