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From the Department of Pathology,* University of Washington, Seattle, Washington; and the Department of Medicine, Division of Nephrology, Vanderbilt University Medical Center, Nashville, Tennessee
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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and macrophage chemoattractant protein-1. This suggested that microglial activation is negatively regulated by EP2 signaling through suppression of prophagocytic cytokine secretion. However, despite this enhancement of Aß phagocytosis, lack of EP2 completely suppressed Aß-activated microglia-mediated paracrine neurotoxicity. These data demonstrate that blockade of microglial EP2 is a highly desirable mechanism for AD therapy that can maximize neuroprotective actions while minimizing bystander damage to neurons.
However, as demonstrated by numerous examples from other organs, activated tissue macrophages need not be exclusively deleterious. In fact, double-transgenic mice that overexpress an AD-causing mutant form of human APP as well as a natural inhibitor of complement C3 suggest that the innate immune response in AD may in part be beneficial by enhancing clearance of Aß peptides from plaques through phagocytosis.10 Thus, an emerging consensus hypothesizes that aggregated Aß stimulates a presumably beneficial microglial phagocytic response while at the same time activating a neurotoxic glial innate immune response; if overwhelming, microglial phagocytosis fails to remove aggregated Aß and the protracted innate immune response becomes neurotoxic.11 This model of AD pathogenesis implies two apparently mutually exclusive therapeutic strategies: stimulate microglia to enhance Aß clearance and suppress microglial activation to dampen bystander damage to neurons.
PGE2, a product derived from arachidonic acid by cyclooxygenase (COX) and specific synthases, is a potent autocrine and paracrine factor that is distinct from other eicosanoid products of COX because of multiple G-protein coupled receptor subtypes, EP1, EP2, EP3, and EP4 that are linked to functionally antagonistic second messenger systems.12 All EP receptor subtypes are expressed on varying cells in brain; microglia express EP1 and EP2.13 Because of this complexity, PGE2 has been conflictingly reported to mediate neurotoxicity and to be neuroprotective, as well as to enhance and suppress macrophage phagocytosis.14-16 Thus, increased PGE2 in the central nervous system, as occurs early in AD,17 may have both pro- and anti-inflammatory actions and may significantly modulate microglial phagocytosis. Here, we tested the hypothesis that ablation of one microglial PGE2 receptor, EP2, may enhance microglial Aß phagocytosis while suppressing bystander damage to neurons from Aß activation of microglia.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Mice homozygous for disruption of the gene that encodes the EP2(EP2–/–) were backcrossed >12 generations to the BALB/c genetic background. BALB/c wild-type (WT) control mice were obtained from Charles River Laboratories (Wilmington, MA). Mice were maintained in a temperature-controlled specific pathogen-free facility with a strict 12-hour light/dark cycle and with free access to food and water. All experiments were performed exactly as approved by the University of Washington Institutional Animal Care and Use Committee. Antibodies 4G8 and 6E10 (specific for Aß1-17 and so does not recognize the p3 fragment of APP) were from Signet Laboratories (Dedham, MA). CD11b was from Serotec (Raleigh, NC). Antibodies against microtubule-associated protein 2 (MAP-2) and neuronal nuclei (NeuN) were from Chemicon (Temecula, CA). Alexa fluorescent-labeled secondary antibodies were from Molecular Probes (Eugene, OR). 4,6-Diamidino-2-phenylindole-containing mounting medium was from Vector Laboratories (Burlingame, CA). Poly-D-lysine was from BD Biosciences (Bedford, MA). Papain and DNase I were from Worthington Biochemical (Lakewood, NJ). Synthetic Aß1-42 was from Bachem (Torrance, CA) and fluorescein-labeled Aß1-42 was from rPeptide (Athens, GA). AH6809, butaprost, and 17-phenyl trinor prostaglandin E2 (PTPE2) were from Cayman (Ann Arbor, MI). SC51089 was from Biomol (Plymouth Meeting, PA). Forskolin and bisindolylmaleimide (BIM) were from Calbiochem (La Jolla, CA). Culture media, heat-inactivated fetal bovine serum, bovine calf serum, and penicillin/streptomycin were from Invitrogen (Carlsbad, CA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise.
Ex Vivo Studies
Frozen AD hippocampus was sliced into 10-µm-thick sections using a cryostat and sections were mounted onto poly-D-lysine-coated coverslips. Sections were stored at –80°C and, to minimize the variation among sections used for WT and EP2–/– microglia, consecutive sections were selected. Primary microglia cultures were derived from cortices of P1 to P3 neonates. Cells were dissociated using enzyme solution containing Dulbecco’s modified Eagle’s medium (DMEM), ethylenediamine tetraacetic acid (0.5 mmol/L), L-cysteine (0.2 mg/ml), papain (15 U/ml), DNase I (200 µg/ml) followed by trituration. Culture medium (DMEM with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin) was changed after 24 hours of initial seeding. Microglia at the 14th day in vitro (DIV) were separated from the underlying astrocytic monolayer by gentle agitation using their differential adhesive properties and were seeded on hippocampal sections at 1 x 105 cells per section in microglial culture medium for 2 hours followed by an additional 48-hour incubation in serum-free DMEM containing penicillin and streptomycin. To determine purity of microglia, 1 x 104 cells were cultured on chambered slides with treatment of 100 nmol/L of aggregated Aß1-42 for 24 hours followed by cytochemistry analysis using a microglial marker, CD11b. Percentage of positive microglia was normalized by 4,6-diamidino-2-phenylindole nuclei counterstaining. From six independent experiments, purity of microglia was 99.4 ± 0.2%. For immunohistochemical analysis, cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and subjected to formic acid (88%) treatment before application of antibodies against CD11b (1:50) and Aß (4G8, 1:200). Mounting medium containing 4,6-diamidino-2-phenylindole was used to label nuclei. Images were randomly selected using Leica confocal/two-photon imaging system. A representative image with a comparable number of microglia between WT and EP2–/– was compared for reduction of Aß plaques. All images were subjected to quantification of CD11b using MetaMorph computer-assisted software. Data were presented as average positive area in arbitrary units per microglia on top of human brain sections. For Western blot analysis, cultures were lysed with 8 mol/L urea containing proteinase inhibitor cocktail. Lysates with equal amount of protein were subjected to 16.5% Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot using 6E10 antibody. Enhanced chemiluminescence was used to reveal Aß species and intensity of the corresponding bands were quantified using the GS-710 imaging densitometer (Bio-Rad, Hercules, CA). Six separate experiments were performed for each analysis.
Microglial Aß Phagocytosis
At DIV 14, microglia were seeded in six-well plates at 1.5 x 105 cells per well in microglial culture medium. After 2 hours of seeding, medium was changed with 1.5 ml of serum-free DMEM containing penicillin and streptomycin. To make aggregated Aß for phagocytosis, Aß was dissolved in 2 mmol/L NaOH and was diluted into 10 µmol/L of stock solution (100x) in PBS followed by incubation at 37°C for 2 hours and monitored by turbidity. For studying signaling pathways involved in Aß phagocytosis, some microglia were pretreated for 1 hour with AH6809 (20 µmol/L), butaprost (1 to 50 µmol/L), SC51089 (10 µmol/L), PTPE2 (20 µmol/L), forskolin (50 µmol/L), or BIM (10 µmol/L) before treatment with aggregated fluorescein-labeled Aß1-42 (100 nmol/L). After 24 hours of incubation of Aß, cells were washed with PBS followed by trypsinization (0.05%) and centrifugation. Cells were then resuspended in PBS and subjected to flow cytometry. Flow cytometer (FACScan; BD Biosciences, San Jose, CA) set at ungated 10,000 cells per measure was used to analyze Aß uptake as an indication of Aß phagocytosis. Data were analyzed using Summit Mo-Flo software (Cytomation, Fort Collins, CO). Phagocytosis was presented as the percentage of mean fluorescein isothiocyanate intensity in WT controls treated with Aß. Some cells were plated onto chambered slides at 1 x 104 for assessing cellular uptake of Aß using a fluorescent microscope (TE200; Nikon, Melville, NY).
Aß-Activated Microglia-Mediated Neurotoxicity
For primary neuronal cultures, embryonic (E16 to E17) cortices from WT mice were extracted, partially digested with proteases, and gently disrupted before plating at 5 x 105 cells per well in poly-D-lysine/laminin (BD Biosciences) coated 24-well plates. One hour after seeding, medium was replaced with neurobasal medium with B-27 supplement, penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (2 mmol/L). Cytosine arabinoside (2.5 µmol/L) was added on the second day. Purity for neuronal cultures was 98 ± 1% as measured by NeuN antibody. DIV 14 primary microglia derived from WT and EP2–/– mice were plated at 1 x 105 per well of WT neurons (DIV 6). To make aggregated Aß for neurotoxicity assay, 1 mg of Aß1-42 was dissolved in 20 µl of dimethyl sulfoxide and was then diluted into PBS to make 500 µmol/L of stock solution followed by 37°C incubation for 24 hours. Freshly prepared, preaggregated Aß at 12 µmol/L was added into co-culture medium containing DMEM, 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin for 24 hours. Cultures were washed twice with PBS and subjected to fixation with 4% paraformaldehyde in PBS at 4°C for 1 hour. Terminal dUTP nick-end labeling (TUNEL) assay (Roche Applied Science, Indianapolis, IN) was performed per the manufacturer’s instructions, followed by fluorescent immunocytochemistry using MAP-2 antibody to identify neurons. Neurotoxicity was presented as percentage of co-positivity for both TUNEL and MAP-2 in all MAP-2-positive neurons.
Cell Count and Levels of Secreted Cytokines
To measure cell proliferation, microglia from WT and EP2–/– were initially seeded at 1 x 105 cells per 24-well culture plate in serum-containing medium. After 2 hours of incubation, some cultures were changed to serum-free conditions. After an additional 24 hours of incubation, cells were washed with PBS and were trypsinized followed by cell count. Six separate experiments were performed to obtain cell count. For cytokine analysis, culture medium from serum-free condition was collected from WT and EP2–/– microglial cultures and were stored at –20°C until measurements were performed. Cytokine/chemokine membrane array (Ray Biotech, Norcross, GA) were used to screen for 32 molecules: interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL12, IL-12p70, IL-13, IL-17, interferon-
, six conserved cytokines (6Ckine), cutaneous T cell-attracting chemokine, eotaxin, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, cytokine-induced neutrophil chemoattractant (KC), leptin, macrophage chemoattractant protein (MCP)-1, MCP-5, macrophage inflammatory protein (MIP)-1, MIP-2, MIP-3ß, RANTES (regulated on activation, normal T cell expressed and secreted), stem cell factor, thymus and activation-regulated chemokine, tissue inhibitors of matrix metalloproteinases-1, tumor necrosis factor-, soluble tumor necrosis factor receptor type I, thrombopoietin, and vascular endothelial cell growth factor. Enzyme-linked immunosorbent assay kits for mouse MCP-1 (Pierce, Rockford, IL), MIP-1 (Biosource Int., Camarillo, CA), and macrophage colony-stimulating factor (Pierce) were used according to the manufacturers’ instructions. Levels of cytokines were obtained at pg per ml of culture medium from 105 microglia cultivated at basal conditions.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We first used a human ex vivo model by incubating primary mouse microglia on top of hippocampal sections from patients who died with AD and participated in the rapid autopsy program at the University of Washington. Confocal images showed abundant Aß-immunoreactive (IR) material in the distribution of senile plaques in an AD tissue section that was incubated with WT microglia (Figure 1A)
and, similar to the results of others, no substantial change from a consecutive tissue section that was incubated without WT microglia (not shown). In contrast, incubation of the next tissue section with EP2–/– microglia (Figure 1B)
markedly reduced Aß-IR material in tissue. Although several possible mechanisms exist to explain the reduction of Aß-IR material in human hippocampus caused by EP2–/– microglia, EP2–/– microglia displayed morphological features of Aß phagocytosis, including pseudopodia engulfing Aß-IR material (arrow in inset of Figure 1B
), that were not present in WT microglia incubated with AD tissue. Moreover, EP2–/– microglia incubated with AD tissue contained Aß-IR material (Figure 1D)
inside phagocytic vacuoles as indicated by intracellular compartments that were IR for CD11b, an indicator for microglial activation (Figure 1E)
.18
In addition, DNA fragments, likely released from necrotic cells in the tissue section, were also seen within these vacuoles (Figure 1F)
. Co-localization among Aß, CD11b, and DNA fragments is shown in Figure 1C
.
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To quantify disappearance of Aß peptides in this ex vivo model, samples were extracted and subjected to Western blot analysis. Aß1-40 and Aß1-42 levels from AD tissue sections were determined from the corresponding band density using densitometry. We found that, similar to the results of others, tissue incubated with WT microglia had Aß1-40 and Aß1-42 levels that were no different from consecutive sections incubated without microglia (not shown). In contrast, tissue sections incubated with EP2–/– microglia had Aß1-40 reduced to 44 ± 5% (P < 0.05, n = 6) and Aß1-42 reduced to 43 ± 4% (P < 0.01, n = 6) after 48 hours compared to consecutive tissue sections incubated with WT microglia (Figure 2, A and B)
; by inspection, minimal clearance of Aß immunoreactivity had occurred by 24 hours. Estimation of CD11b IR per microglial cell incubated with human brain tissue slices was performed using confocal images from WT (Figure 2C)
and EP2–/– (Figure 2D)
. Data showed that CD11b IR in WT or EP2–/– microglia was 23 ± 2 arbitrary fluorescence units (n = 5) or 110 ± 7 arbitrary fluorescence units (n = 10, P < 0.01), respectively (Figure 2E)
; these data associate reduction of Aß burden in AD brain section with a standard measure of microglial activation. Although the total number of microglia present on AD tissue were equivalent regardless of genotype at the end of incubation (159 ± 17, n = 5, WT microglia in x250 fields; and 165 ± 22, n = 10, EP2–/– microglia in x250 fields), they were not similarly distributed; EP2–/– microglia tended to aggregate around Aß-IR plaques whereas WT microglia were more evenly dispersed across the tissue section, perhaps suggesting enhanced chemoattaction in EP2–/– microglia. In combination, these observations showed that EP2–/– microglia had enhanced phagocytic activity toward fibrillar Aß peptides and reduced Aß peptide tissue burden in human hippocampal slices much more effectively than WT microglia. Although the precise mechanisms of reduced Aß tissue in these experiments have not been determined, we speculate, as have most others5,19,20
that this occurs by degradation of phagocytosed protein.
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To characterize better the enhanced Aß1-42 phagocytosis by EP2–/– microglia, primary cultures of microglia from WT and EP2–/– mice were incubated with aggregated Aß1-42 labeled with fluorescein and analyzed by microscopy and flow cytometry. Consonant with our ex vivo studies described above, EP2–/– microglia (Figure 3B)
had greater Aß1-42 uptake compared to WT (Figure 3A)
. Some EP2–/– microglia displayed phagocytosed Aß that exceeded that observed in WT microglia (arrow in Figure 3B
), whereas other EP2–/– microglia appeared similar to their WT counterparts, suggesting a heterogeneous population of EP2–/– microglia with a subpopulation of cells with enhanced phagocytic properties for Aß1-42. A representative scatter plot from flow cytometric analysis showed that granularity and size were not different between WT and EP2–/– microglia (Figure 3C)
. Flow cytometric data (Figure 3D)
showed a shift toward higher fluorescent intensity in EP2–/– microglia compared to WT after incubation with fluorescein-labeled Aß1-42. Using a cutoff of the peak value for EP2–/– microglia, the percentage of tested cells with high-fluorescent intensity in WT (Figure 3E)
or EP2–/– microglia (Figure 3F)
was 12 ± 1% (n = 10) or 38 ± 1% (n = 10), respectively (P < 0.001). However, there was overlap between microglia of the two genotypes at low Aß1-42 uptake (Figure 3D)
. These data support the hypothesis that a subpopulation of EP2–/– microglia has enhanced phagocytic properties for Aß1-42 and suggest that ablation of EP2–/– relieves an inhibitory mechanism thereby leading to a greater proportion of cells with heightened phagocytic activity for Aß1-42.
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We next investigated the signaling events that underlie augmented Aß1-42 phagocytic activity in EP2–/– microglia using the same flow cytometric technique (Figure 4)
. Phagocytosis of Aß1-42 was 2.5- to 3-fold greater in EP2–/– microglia compared to WT (P < 0.0001). WT and EP2–/– microglia had 36 ± 4% (P < 0.001) and 35 ± 4% (P < 0.001) of WT basal Aß1-42 uptake when incubated with the PKC inhibitor BIM at 10 µmol/L, suggesting that all of the enhanced Aß1-42 phagocytosis observed in EP2–/– microglia and part of basal Aß1-42 phagocytosis in WT microglia are PKC-dependent. A PKA inhibitor (H89) was used in WT microglia and a PKA activator (forskolin) was used in EP2–/– microglia to determine whether this signaling pathway was contributing to the enhanced Aß1-42 uptake. H89 did not enhance Aß1-42 uptake in WT microglia and so did not replicate the EP2–/– phenotype, and forskolin did not reverse the enhanced Aß1-42 phagocytosis observed in EP2–/– microglia. We next investigated the effects of the relatively selective EP1 antagonist, SC51089 at 10 µmol/L, or EP1 agonist, PTPE2 at 20 µmol/L, on Aß1-42 phagocytosis by microglia. We found that both drugs were without effect in either WT or EP2–/– microglia, suggesting that EP1 activation or blockade does not significantly alter microglial Aß1-42 phagocytosis. Finally, butaprost, a relatively selective EP2 agonist, did not alter Aß1-42 uptake in WT microglia (98 ± 3% at 1 µmol/L, 104 ± 2% at 10 µmol/L, and 92 ± 2% at 50 µmol/L; n = 6 for each condition), suggesting that stimulation of EP2 does not have the opposing effect of ablating EP2. These data indicated that basal Aß uptake in WT microglia is EP2-independent and EP2-mediated suppression of microglial Aß1-42 phagocytosis is primarily, if not completely, PKC-dependent.
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As seen in Figure 1
, enhanced Aß peptide phagocytosis by EP2–/– microglia in human tissue sections was associated with morphological features associated with activation. Although this may result from stimulation of microglia by tissue components, it also may result, at least in part, from constitutive activation of EP2–/– microglia; here we investigated the latter (Table 1)
. One feature of microglial activation is increased proliferation. We investigated this by counting primary microglia after 24 hours in culture after plating under identical conditions and observed no difference between WT and EP2–/– microglia. Another indicator of activation is cytokine and chemokine secretion. Conditioned culture medium was screened for secretion of 32 cytokines and chemokines using a membrane array that has the advantage of simultaneously measuring multiple molecules but the disadvantage of relative insensitivity. Only two chemokines, MCP-1 and MIP-1, were sufficiently elevated in conditioned medium from EP2–/– microglial cultures to be detected by cytokine array, whereas WT microglia had no detectable cytokines or chemokines when simultaneously assayed (data not shown). Data from enzyme-linked immunosorbent assay confirmed that MCP-1 and MIP-1 were significantly greater in EP2–/– than WT-conditioned medium (P < 0.001) (Table 1)
. Macrophage colony-stimulating factor, reported to increase microglial phagocytosis of Aß,21
was not detected by our membrane array or by enzyme-linked immunosorbent assay (limit of detection of 5 pg/ml) of conditioned medium from WT or EP2–/– microglia. Thus, even without specific exogenous stimuli, EP2–/– microglia displayed some features of an activated phenotype with increased secretion of MCP-1 and MIP-1 compared to WT microglia, but not increased proliferation by cell count. Moreover, these data indicate that at least some chemoattractant chemokines, namely MCP-1 and MIP-1, are regulated by EP2 signaling.
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Although enhanced Aß1-42 phagocytosis and reduction of Aß peptide burden in AD tissue seen with EP2–/– microglia is desirable, if it comes at the cost of increased activation with increased neurotoxicity, then it is not clear if this is a true benefit to patients with AD. To test whether Aß-activated EP2–/– microglia had enhanced paracrine neurotoxicity, we used primary cultures of WT neurons and microglia from either WT or EP2–/– mice. The final dimethyl sulfoxide concentration in these experiments was 0.1% and was nontoxic to primary microglia or neurons. As expected from the results of others,22-24
exposure of neurons alone to aggregated Aß1-42 was associated with dendrite atrophy (Figure 5B)
and decreased neuron number as assessed by MAP-2 IR (Table 2)
. Even greater Aß1-42-initiated dendrite atrophy and neuron loss was observed in cultures that combined neurons and WT microglia (Figure 5C
and Table 2
). These results were confirmed in the remaining MAP-2 IR cells that showed increased TUNEL positivity after exposure to Aß1-42 and still further increased TUNEL positivity in MAP-2 IR cells in cultures containing neurons and WT microglia (Table 2)
. Strikingly, cultures that contained neurons and EP2–/– microglia showed no evidence of neurotoxicity by either of these endpoints or morphological assessment (Table 2
and Figure 5D
). Indeed, by one measure, TUNEL positivity, co-culture with EP2–/– microglia was associated with apparent neurotrophism. The lack of Aß1-42-mediated neurotoxicity in EP2–/– microglia/WT neuron co-cultures strongly suggests that, although Aß1-42 can be directly toxic to isolated neurons in culture, when combined with microglia the major neurotoxins are paracrine factors secreted by Aß1-42-activated microglia. WT microglial activation by Aß yielded the expected large increase in COX-2 and iNOS as determined by Western blot; primary and secondary products of both enzymes can cause neurotoxicity.25-27
Activation of EP2–/– microglia under identical conditions resulted in COX-2 and iNOS induction that were 16 ± 1% and 43 ± 4%, respectively, of WT microglia (n = 3 for each, P < 0.01 for both). Although the mechanisms by which microglia may damage neurons are more complex than simply COX-2 and iNOS products, these data indicate at least two mechanisms by which Aß-activated EP2–/– microglia may be less neurotoxic.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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As a justification for Aß vaccination trials, others have shown that culturing WT rodent or human microglia on human AD brain sections that had been incubated with anti-Aß antibodies results in clearance of Aß from tissue and appearance of Aß-IR material in intracellular phagosomes. Presumably this anti-Aß antibody-enhanced microglial phagocytosis of tissue Aß is because of opsonization and Fc receptor-dependent phagocytosis in ex vivo systems,28-30 although this has been challenged as the mechanism of action of Aß vaccination in vivo in which non-Fc receptor-dependent phagocytosis is also involved.20,31 It is important to note that without incubation of tissue with anti-Aß antibodies and presumed opsonization of tissue Aß, WT microglia do not phagocytose Aß or clear Aß from human tissue sections ex vivo.29 To our knowledge, ours is the first demonstration of genetically altered microglia achieving similar Aß clearance from human tissue sections without involvement of antibody binding as anti-Aß opsonization followed by WT microglia reported by others.
Recently, others have shown that EP2 on rodent alveolar macrophages suppresses Fc receptor-mediated phagocytosis of bacterial pathogens.14
Importantly, EP2-mediated suppression of Fc receptor-mediated phagocytosis by alveolar macrophages was cAMP-dependent and the suppressed phagocytic phenotype was restored in EP2–/– macrophages by treatment with forskolin; the participation of EP2 in Fc receptor-independent macrophage phagocytosis is not yet reported. Similar to these investigators, we also observed an EP2-dependent suppressive effect on microglial Aß phagocytosis; however, this was dependent on PKC activation and was not modified by drugs that activate or inhibit adenylate cyclase. Importantly, we observed that inhibition of PKC in EP2–/– microglia restored the suppressed phagocytic phenotype in EP2–/– microglia to WT levels, strongly suggesting that postreceptor signaling cascades were intact in EP2–/– microglia. Because Aß phagocytosis is thought to be, at least in part, opsonin-independent and mediated by a group of receptors including formyl peptide receptor, scavenger receptors, and receptor for advanced glycation end-products,19
these data raise the possibility that, in contrast to Fc receptor-dependent phagocytosis, EP2-mediated suppression of Fc receptor-independent phagocytosis may be mediated by PKC-dependent mechanisms. The exact effectors responsible for this novel mechanism underlying negative regulation of Aß phagocytosis through EP2 signaling are not entirely clear; however, the mechanism is likely related, at least in part, to increased secretion of prophagocytic ß chemokines, such as MIP-1 and MCP-1. Because ß chemokine signaling during inflammation requires PKC activity,32,33
one possibility is that downstream effectors of EP2 activation suppress phagocytosis by inhibiting ß chemokine signaling, activity of PKC isoforms, or both.
If enhanced Aß phagocytosis resulted from generalized activation of microglia with correspondingly enhanced innate immune response and increased bystander damage to neurons, then the utility of microglial EP2 as a therapeutic target would be unclear. Previously, we have observed in a murine model of activated cerebral innate immune response from intracerebroventricular injection of lipopolysaccharide that neuronal oxidative damage is completely suppressed in EP2–/– mice;34
however, interpretation of these data are confounded by microglial and neuronal expression of EP2. Others have shown that peroxisome proliferator-activated receptor (PPAR) agonists can suppress EP2 expression in lung carcinoma cell lines35
and that PPAR agonists also suppress Aß-stimulated microglial paracrine neurotoxicity.36
Therefore, EP2 may be critical in microglia-mediated neurotoxicity. Here we specifically determined that ablation of microglial EP2 only, without any genetic alteration to neurons, completely suppressed Aß1-42-mediated neurotoxicity. Moreover, as suggested by results from the TUNEL assay, EP2–/– microglia may even be mildly neurotrophic. Thus, EP2–/– microglia possess the highly desirable complementary traits of enhanced Aß phagocytosis without enhanced bystander damage to neurons. Given the complications of Aß vaccination,37
these results point to suppression of microglial EP2 as a perhaps safer but similarly efficacious means of reducing Aß burden in human brain.
In conclusion, our study provides new insights into the central role of EP2 in regulation of microglial Aß phagocytosis and Aß-stimulated microglia-mediated paracrine neurotoxicity. Critically, we showed that ablation of microglial EP2 achieved the highly desirable combination of enhancing Aß phagocytosis while at the same time completely suppressing bystander damage to neurons. If these results can be extended to human microglia, then these data indicate blockade of microglial EP2 signaling as a means of maximizing beneficial while minimizing deleterious effects of microglia in AD brain.
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Footnotes |
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Supported by the National Institutes of Health (grants AG24011, AG05144, and AG05136).
Accepted for publication December 20, 2004.
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References |
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–/– knock-out mice. J Neurosci 2003, 23:8532-8538 agonists. J Neurosci 2000, 20:558-567 ligands inhibits human lung carcinoma cell growth. Biochem Biophys Res Commun 2004, 314:1093-1099[Medline]
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