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Endocrinology Vol. 148, No. 7 3383-3390
Copyright © 2007 by The Endocrine Society

Androgen-Mediated Immune Function Is Altered by the Apolipoprotein E Gene

Candice M. Brown, Qing Xu, Nobutaka Okhubo, Michael P. Vitek and Carol A. Colton

Division of Neurology, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Carol A. Colton, Ph.D., Division of Neurology, Duke University Medical Center, Box 2900, Durham, North Carolina 27710. E-mail: glia01{at}aol.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens, like estrogens, have been linked to neuroprotective effects in the brain and to the improvement of cognitive function. Part of this effect may be due to the action of androgens on the innate immune response. We have examined the action of dihydrotestosterone (DHT) and testosterone on immune activation in primary cultures of microglia, the central nervous system macrophage. Our data indicate that DHT acts as an antiinflammatory agent and depresses both nitric oxide and TNF{alpha} production in a dose-dependent fashion. However, testosterone treatment of microglia and peritoneal macrophages increased supernatant nitrite levels, indicative of a proinflammatory effect. Because the apolipoprotein E (APOE) genotype also dramatically impacts macrophage function and has been linked to neurodegenerative disease, we compared the effects of APOE genotype on androgen-mediated regulation of inflammation using targeted replacement mice expressing only the human APOE3 or human APOE4 gene. Our data show that the antiinflammatory activity of DHT is significantly reduced in APOE4 targeted replacement mice compared to APOE3 mice. The effect was not due to an APOE isoform-specific change in androgen receptor mRNA and protein expression. Rather, innate immune signaling pathways regulated by androgens are altered in the APOE4 microglia. Compared to APOE3 microglia, DHT treatment did not reduce the phosphorylation of p38 MAPK or p54/p56 Janus kinase in APOE4 mice. Thus, our data suggest that DHT modulation of kinase activity is altered in microglia from mice expressing an APOE4 genotype and may impact androgen treatment therapies in individuals with an APOE4 genotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GENDER-BASED DIFFERENCES IN neurodegenerative diseases such as Alzheimer’s disease (AD) have been an intensely debated public health issue, particularly because of the potential usefulness of hormone replacement therapy as a treatment for memory loss and cognitive dysfunction in aging individuals. Studies on the lack of estrogen and on estrogen replacement in aging women have dominated the field due to a multiplicity of studies elucidating the direct neuroprotective role of estrogen and its antiinflammatory properties (1, 2, 3, 4). However, a similar case could be built for androgens because the beneficial effects of androgen treatment on neurons generally parallel that of estrogens (5).

Androgens, like estrogens, play important roles in the brain of both males and females. For example, testosterone prevents apoptosis and enhances cell survival in primary neuronal cultures derived from human fetal brain (6). Exogenous testosterone enhances motor neuron repair after axonal damage, by both direct neuronal and indirect glial mechanisms (7). In addition, testosterone prevents {tau} hyperphosphorylation induced by heat-shock mediated glycogen synthase kinase 3ß activation, reduces the neuronal secretion of ß-amyloid peptides, and protects against ß-amyloid-mediated neuronal toxicity (8, 9, 10, 11, 12). Recent studies have also suggested that androgens improve cognitive function in male rats, male mice, and in normal human males (5, 13). However, contradictory data exist that find that testosterone either has no effect or is detrimental to learning and memory (14, 15, 16). In general, testosterone is thought to be beneficial to neurons (17, 18), and a reduction in testosterone, as observed in aging males, is associated with central nervous system (CNS) dysfunction including AD (19, 20, 21, 22, 23).

Because neuroinflammation is observed in most, if not all, neurodegenerative diseases, the effect of androgens on inflammation also becomes a critical aspect of the response of the brain in males to injury and disease. Expression of androgen receptors (AR) on microglia, the brain macrophage, increases after injury and indicates that the innate immune cells of the brain may be modulated by androgens (24). Studies on peripheral macrophages clearly demonstrate that testosterone alters immune function in a complex manner. Testosterone can be both proinflammatory and antiinflammatory. For example, wound healing is impaired in males, especially the elderly, and has been directly linked to a proinflammatory action of testosterone on tissue macrophages in the skin (25). The opposite response, i.e. depression of immune function, is observed in testosterone-treated animals after trauma-hemorrhage and in relapsing experimental autoimmune encephalomyelitis (26, 27). Ashcroft and Mills (25) have shown that castration increased macrophage-mediated damage at sites of injury in the skin and suggests an antiinflammatory role for testosterone. Testosterone also inhibits inducible nitric oxide (NO) synthase and NO production in RAW 264.7 cells, a mouse macrophagic cell line (28). Overall, no clear, unequivocal effect of testosterone on innate immunity has been demonstrated.

An interrelationship between testosterone levels, neurodegenerative diseases such as AD, and the apolipoprotein E (APOE) 4 genotype, a known genetic risk factor for AD (29), has recently emerged (23, 30, 31). The APOE genotype contributes to the age-related changes in androgen levels and AR expression in men (23, 31, 32). Hogervorst et al. (30) have shown that normal men who are APOE4 carriers have a lower testosterone level than men who are not APOE4 carriers. However, this relationship does not include male APOE4 carriers diagnosed with AD. In this case, although a decrease in testosterone level is observed, it does not reach statistically significant values. In addition, males who are homozygous for the APOE4 allele demonstrate a significant downward shift in scores for episodic memory compared with male APOE4 heterozygotes (33). A relationship between learning and memory deficits and APOE genotype has also been observed in mice expressing human APOE3 or human APOE4 under a neuron-directed promoter (neuronal-specific enolase) (34).

Our previous findings on targeted replacement APOE3 and APOE4 mice suggest that the inflammatory response may be different in male and female mice when the APOE4 gene is present (35, 36). Thus, we have examined the interaction between androgens and APOE genotype in immune activation of murine microglia and of adult murine peritoneal macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experiments were conducted in compliance with the National Institutes of Health guidelines and have been approved by the Duke University Institutional Animal Care and Use Committee. Animals were housed under a standard 14-h light, 10-h dark cycle and received food and water ad libitum. Experiments were performed using homozygous APOE targeted replacement mice. These mice contain a targeted insertion of exons 2–4 of the human APOE3 or APOE4 genes that replace the corresponding genomic DNA at the mouse APOE locus (37). Thus, only human apoE3 or human apoE4, the protein products of the human APOE3 and APOE4 genes, respectively, are produced.

Gonadectomy (GDX)
Adult male APOE3 (33 ± 3 wk) and APOE4 (26 ± 4 wk) mice were anesthetized using ip sodium pentobarbital (40–70 mg/kg) and a sterile field prepared. A 2-cm ventral midline incision was made in the scrotum, and the skin was retracted to expose the tunica. After piercing the tunica, the opening was stretched with blunt forceps, and gentle pressure on the pelvis was used to isolate the testis. The spermatic artery was clamped and cauterized, and the testis was removed. The epididymis, deferential vessels, and ductus were then replaced in the tunica and the incision was closed using wound clips. All animals were returned to their cage and were housed under a standard light, dark cycle and received food and water ad libitum.

In one set of experiments, a sterile 60-d time-release pellet containing 12.5 mg testosterone (Innovative Research of America, Sarasota FL) was implanted sc into GDX males 1 d after castration. This group was designated as gonadectomized + testosterone pellet (GDX + test. pellet). Pellets were designed to deliver approximately 1200 ng/ml testosterone. Mice were treated for 4 wk before collection of blood samples and peritoneal macrophage isolation.

Before harvesting peritoneal macrophages, blood was collected by cardiac puncture to determine the serum testosterone concentrations. Testosterone levels in the serum were measured using the testosterone 125I RIA Kit from ICN Diagnostics (Costa Mesa, CA) per the manufacturer’s instructions. Before measurement, each blood sample was extracted using diethyl ether (4 ml diethyl ether; 0.300 ml saline/serum sample), nitrogen evaporated at 37 C and reconstituted into diluent buffer provided from the kit. Average circulating testosterone levels in intact male APOE3 mice were not significantly different from intact male APOE4 mice (890 ± 220 and 880 ± 200 pg/ml, respectively). Serum testosterone levels were below the detectable range (<10 pg/ml) in GDX males. Values of serum testosterone in the GDX + test. pellet group were 700 ± 300 pg/ml for APOE3 males and 680 ± 110 pg/ml for APOE4 males.

Peritoneal macrophage isolation and culture
Peritoneal macrophages were elicited by ip injection of each mouse with 5 mM sodium periodate for 72 h and were isolated as previously described (36). Lavage fluid containing cells was removed from the peritoneum, and the fluids containing cells from two to three mice of the same genotype were pooled. The cells were pelleted by centrifugation at and then resuspended into phenol-red-free, serum-free media (high glucose DMEM containing 2 mM glutamine and 50 µg/ml gentamycin). Cells were counted, plated directly into 96-well plates, and cultured for 2–3 d in a humidified 5% CO2, 95% O2 air atmosphere. During incubation, the macrophages attach to the plastic of the culture plate and spread, resembling typical tissue macrophages.

Microglial cultures
Enriched microglial cultures were prepared from postnatal d 0–2 APOE3 and APOE4 pups as previously described (38). Briefly, whole brains were removed and placed into sterile 1x PBS (1x PBS) containing 100 µg/ml penicillin/streptomycin and 0.5% fungizone (Invitrogen, Rockville, MD). Meninges were removed and cortices were dissociated using the Papain Dissociation System (Worthington Biochemicals, Lakewood, NJ) per the manufacturer’s instructions. Cells were then plated into T75 tissue culture flasks (three to five brains per flask per litter group) and were grown at 37 C in high glucose DMEM containing 100 µg/ml penicillin/streptomycin, 2 mM glutamine, and 10% fetal bovine serum (FBS). The resulting mixed glial culture was grown for 3–5 d, at which time the culture media was replaced with fresh growth media containing 10% horse serum in place of FBS. After an additional 3–5 d, loosely adherent cells (primarily microglia) were removed from the culture by shaking on a rotary shaker (240 rpm) for 2 h. The cells were pelleted by centrifugation (1000 x g; 10 min) and resuspended into phenol red-free, serum-free medium. The cells were then plated into 96-well dishes for measurement of supernatant nitrite or TNF{alpha} or into 12-well dishes for RNA determinations and were cultured for an additional 48 h in serum-free, phenol red-free media. Once plated, serum withdrawal or extended culture in serum-free medium does not adversely affect microglial survival (data not shown) (36).

Immune activation in vitro
Microglial cultures were pretreated for 15 h (overnight) with dihydrotestosterone (DHT), a nonaromatizable form of testosterone, before immune activation. Stock solutions of 1 mM DHT or testosterone (Sigma, St. Louis, MO) were prepared in sterile DMSO and diluted to a final concentration in the nanomolar range in serum-free, phenol red-free microglial media for use in the experimental protocols. DMSO concentrations in all final media were less than 0.001% and considered to be negligible. For detection of supernatant nitrite and TNF{alpha}, DHT-treated and untreated macrophages were immune activated for 20–24 h using varying concentrations of recombinant mouse interferon-{gamma} (IFN{gamma}) (100 U/ml; 2.5 ng/ml) and/or lipopolysaccharide (LPS), a bacterial endotoxin prepared from Escherichia coli serotype O55:B5 (all from Sigma) (100 ng/ml) diluted into phenol red-free, serum-free media. To maintain exposure of the pretreated cells to DHT, the same concentration of DHT that was used in the pretreatment protocol was also added to the media containing immune activators. For quantitative analysis of mRNA, DHT-treated or untreated cells were immune activated for 5 h using the same activating agents as above. For analysis of kinase activation, cells were pretreated with DHT and 1% charcoal-stripped FBS (Hyclone, Logan, UT) for 15 h and immune activated with LPS-IFN{gamma} for 30 min.

Measurement of nitrite and protein
The supernatant levels of nitrite, the stable oxidation product of NO in biological solutions, were used to determine macrophage NO production and was measured by chemiluminescence using a Sievers 280 Nitric Oxide Analyzer (Sievers, Boulder, CO). Standard curves were prepared from sodium nitrite diluted into the treatment media. Total protein (micrograms per well) was measured using the BCA method (Pierce, Rockford, IL) with BSA (micrograms per milliliter) as standard. Nitrite levels in each well were normalized to total micrograms of protein, and data points represent the average nitrite value per microgram of protein ± SEM.

Measurement of TNF{alpha}
Supernatant levels of TNF{alpha} were determined by ELISA per the manufacturer’s instructions (BioSource International, Camarillo, CA). TMB-One (Promega, Madison, WI) was used for substrate color development, and the reaction was stopped using 1 M H2SO4. Absorbance was measured at 450 nM using a Molecular Devices microplate reader (Molecular Devices, Sunnydale, CA). TNF{alpha} values were normalized to micrograms of protein and presented as picograms of TNF{alpha} per microgram of protein. Data points represent the average TNF{alpha} per microgram of protein values ± SEM.

Immunoblots
After pretreatment for 15 h with 0, 1, 5, or 10 nM DHT, microglia were immune stimulated with 10 ng/ml of LPS plus 10 U/ml recombinant murine IFN{gamma} for 30 min. Western blot analysis was performed to determine the expression of AR or the phosphorylated and total forms of three members of the MAPK family: p38, Janus kinase (JNK) p54/p46, and ERK1/2. Cells cultured in 12-well plates were washed with PBS, and lysed with 200 µl of lysis buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1% SDS, and 100 mM NaCl) containing a protease inhibitor cocktail (Roche, Indianapolis, IN). Samples were boiled for 5 min, separated by electrophoresis on 4–20% NOVEX Tris-Glycine gels (Invitrogen, Carlsbad, CA), and transferred onto Immobilon membrane (Millipore Corp., Bedford, MA). The membrane was incubated in blocking buffer (1x PBS, 5% nonfat dried milk) for 1 h at room temperature and then probed with a primary antibody in blocking buffer overnight at 4 C. Anti-ERK antibody (p44/42 MAP Kinase; no. 9102), anti-phospho-ERK antibody (Thr202/Tyr204; no. 9101), anti-JNK antibody (no. 9252), anti-phospho-JNK antibody (Thr183/Try185; no. 9255), anti-p38 MAP Kinase antibody (no. 9212) and anti-phospho-p38 MAP Kinase antibody (Thr180/Tyr182; no. 9211) were purchased from Cell Signaling Technology (Beverly, MA). All aforementioned primary antibodies were used at a 1:1000 dilution. Antibody against human AR (mouse monoclonal; ab441) was purchased from AbCam (Cambridge, MA) and was used at a dilution of 1:100. After three washes in PBS containing 0.1% Tween 20, blots were probed with the secondary antibody in blocking buffer for 1 h at room temperature, and washed again in PBS containing 0.1% Tween 20. Detection of signal was performed with an enhanced chemiluminescence detection kit (Amersham GE, Piscataway, NJ). Band densities were measured and analyzed using Kodak Molecular Imaging Software version 4.0 (Eastman Kodak Company, Rochester, NY), and the ratio of phosphorylated kinase to total kinase was determined. Values are presented as the average relative density and represent the ratio of total phosphoprotein to total protein.

RNA extraction and preparation/quantitative RT-PCR
RNA was extracted from microglial cultures or from the cortex of APOE3 and APOE4 mice using the RNAEasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. RNA was quantified using a Beckman DV530 Life Science UV/Visible Spectrophotometer (Beckman-Coulter, Fullerton, CA) and was reverse transcribed to cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) with MultiScribe reverse transcriptase and random primers. mRNA expression levels were determined by quantitative PCR. Essentially, qPCR were performed using 100 ng cDNA per reaction with a ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) and TaqMan Assays-on-Demand Gene Expression primer/probe sets and the Taqman Universal PCR master mix (all from Applied Biosystems) for the murine AR gene (GenBank, NM_ 013476.2; Assay-on-Demand ID, Mm00442688_m1), estrogen receptor {alpha} (ESR1) (GenBank,: NM_007956; Assay-on-Demand ID, Mm00433149_m1); estrogen receptor ß (ESR2) (GenBank, NM_010157; Assay-on-Demand ID, Mm00599819_m1); aromatase (GenBank, NM_007810; Assay on Demand ID, Mm00484049_m1) and eukaryotic 18S, a standard housekeeping gene (GenBank, X03205; Assay-on-Demand ID, Hs99999901_s1). Relative mRNA quantitation was calculated using the 2–{Delta}{Delta}CT method (39) by normalizing the value of the target gene for each sample to its endogenous 18S control value, and then normalizing these values to a baseline sample, designated as the calibrator. In these experiments, the APOE3 untreated microglia sample served as the calibrator for each gene.

Statistical analysis
For microglia and macrophage cultures, average values (±SEM) for each treatment condition were compared between similarly treated APOE3 or APOE4 cultures. Samples were assayed from at least three different litter groups (where a litter group is defined as those cells cultured from different mouse litters; three to five mouse brains were pooled per litter group). Cells from each litter group were subcultured by plating into separate culture dish wells. A minimum of duplicate wells were analyzed per experimental paradigm for each of three or more litter groups. Peritoneal macrophages were used in a similar manner as microglia with the exception that macrophages were harvested and pooled from two to three adult mice. Data were analyzed using two-way ANOVA with Bonferroni’s post test where appropriate, using the Prism 4.0 software package (Graphpad, San Diego, CA). In cases where the number of samples analyzed were different, the Prism program used an unweighted means analysis. Significance was set at P ≤ 0.05 in all cases.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effect of varying doses of DHT, the nonaromatizable form of testosterone, on immune activation in primary cultures of microglia was examined by analyzing changes in TNF{alpha} and nitrite production. Microglia were pretreated with physiological levels of DHT (1–10 nM) for 15 h in serum-free, phenol-red-free media and then immune activated with the combination of LPS and IFN{gamma} (LPS + IFN{gamma}). These immune agents are well known to elicit a proinflammatory response in macrophages that includes the release of TNF{alpha} and NO (with the subsequent formation of nitrite) into the cell supernatants (40). As shown in Fig. 1AGo, NO production, as measured by supernatant nitrite levels, was decreased by DHT in a dose-dependent fashion in APOE3 microglia. In contrast, LPS + IFN{gamma}-activated APOE4 microglia did not respond to DHT within the concentration range studied. Using two-way ANOVA analysis, a significant interaction was detected between APOE genotype and DHT treatment (P < 0.04; F(3,99) = 2.94), indicating that the APOE genotype influenced the response to DHT. A similar effect was observed for TNF{alpha} production. In Fig. 1BGo DHT treatment of microglia derived from APOE3 mice significantly decreased the level of TNF{alpha} produced during immune stimulation in a dose-dependent manner. Again, no significant change in TNF{alpha} production was observed with DHT in similarly treated APOE4 microglia. A significant interaction between APOE genotype and DHT treatment was also observed for microglial TNF{alpha} production (P = 0.048; F(3,28) = 2.98).


Figure 1
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  		FIG. 1. APOE genotype-specific action of DHT on immune-activated microglia. Cultured microglia from either APOE3 or APOE4 mice were pretreated with varying doses of DHT (nanomolar) for 15 h and then immune activated by the addition of 100 U/ml IFN{gamma} and 100 ng/ml LPS for an additional 24 h. A, Supernatant nitrite levels were used to detect NO production in DHT-treated APOE3 and APOE4 microglial cultures. Data represent the mean (±SEM) nitrite levels (nanomoles per microgram of protein). A significant interaction between APOE genotype and DHT was observed (P = 0.037). ***, P < 0.001 for DHT treatment compared with LPS + IFN{gamma} alone (0 DHT); ##, P < 0.01 for APOE4 compared with APOE3. B, Average (±SEM) supernatant TNF{alpha} concentrations (picograms per microgram of protein) from immune-activated APOE3 and APOE4 microglia were determined using an ELISA. A significant interaction was observed (P = 0.048) between genotype and DHT treatment. *, P < 0.05 for DHT treatment compared with LPS + IFN{gamma} alone; #, P < 0.05; ###, P < 0.001 for APOE4 compared with APOE3.

 
To determine whether APOE3 and APOE4 microglia responded to other androgens in addition to DHT, we also examined the effect of testosterone on immune activation using a similar treatment paradigm. As shown in Fig. 2Go, testosterone treatment in APOE3 microglia produced a significant increase in supernatant nitrite levels at 1 nM but returned to control values at 10 nM. Treatment of APOE4 microglia with testosterone also produced an increase in nitrite which returned to control levels at higher doses. These data demonstrate that both APOE3 and APOE4 microglia respond similarly to testosterone. However, the response is different from the antiinflammatory action of DHT. Androgens have been reported to elicit both antiinflammatory and proinflammatory actions as part of an innate immune response (25, 28, 41). Our data confirm these findings and further support the fact that androgen interaction with microglia can promote (testosterone) or reduce (DHT) inflammatory signals (25, 26, 42).


Figure 2
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  		FIG. 2. Effect of testosterone on microglial function. APOE3 and APOE4 microglia were pretreated with 1–10 nM testosterone, immune activated with LPS + IFN{gamma}, and the average supernatant nitrite level (±SEM) was measured. **, P < 0.01, ***, P < 0.001 for testosterone treated compared with LPS + IFN{gamma} alone (0 testosterone).

 
Because microglia are derived from brains of neonatal, prepubertal animals, we examined the effect of APOE genotype on peritoneal macrophage function from adult APOE3 and APOE4 GDX male mice. Serum levels of testosterone were negligible (<10 pg/ml) in both the APOE3 and APOE4 male mice after GDX. To test the effect of androgens, testosterone was replaced in vivo using a testosterone slow-release pellet implanted sc. Serum levels of testosterone were not significantly different between APOE3 and APOE4 male mice after testosterone replacement and were within a physiological range. Thus, differences in circulating testosterone levels between APOE3 and APOE4 mice were removed as a potential confounding factor. For this experiment, peritoneal macrophages were isolated from APOE3 and APOE4 GDX and GDX + test. pellet adult male mice and then immune activated with LPS + IFN{gamma}. Similarly to microglia, we measured supernatant levels of nitrite and TNF{alpha} as markers of an inflammatory response (Fig. 3Go, A and B). Nitrite levels were significantly higher in culture supernatants of LPS + IFN{gamma}-activated macrophages from adult APOE3 mice treated by endogenous replacement of testosterone (GDX + test. pellet) compared with untreated APOE3 GDX mice (GDX-only). However, replacement of testosterone in adult APOE4 mice did not produce a significant change in peritoneal macrophage nitrite levels. Using two-way ANOVA, a significant interaction (P ≤ 0.0001; F(1,133) = 23.88) was observed between genotype and the peritoneal macrophage response to treatment, suggesting that the APOE genotype strongly influenced the effect of testosterone on NO production. A significant interaction between APOE genotype and TNF{alpha} production was also observed (P = 0.0064; F(1,80) = 7.8). However, in this case, the effect of endogenous testosterone was associated with depression of TNF{alpha} production in APOE3 peritoneal macrophages. In contrast, TNF{alpha} production by macrophages derived from APOE4 GDX + test. Pellet mice was not different from macrophages derived from untreated APOE4 GDX-only mice, suggesting that the responsiveness of APOE4 macrophages is altered compared with APOE3 macrophages.


Figure 3
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  		FIG. 3. Adult peritoneal macrophages from APOE4 mice also demonstrate decreased responsiveness to androgens. LPS + IFN{gamma}-activated adult peritoneal macrophages from gonadectomized APOE3 or APOE4 mice were treated with testosterone in vivo by replacing testosterone via a sc pellet (GDX + test. pellet). The resultant supernatant nitrite and TNF{alpha} levels were then compared with the nitrite and TNF{alpha} levels produced by LPS + IFN{gamma}-activated peritoneal macrophages from untreated GDX mice. A, A significant interaction (P < 0.0001) between APOE genotype and testosterone replacement was observed for nitrite production. ***, P < 0.001 for GDX + test. pellet compared with GDX alone; ###, P < 0.001 for APOE4 compared with APOE3. B, A significant interaction (P = 0.0064) between APOE genotype and testosterone replacement was also observed for TNF{alpha} production. ***, P < 0.001 for GDX + test. pellet compared with GDX alone; #, P < 0.05 for APOE4 compared with APOE3.

 
Androgens exert their effects on cell functions by interacting with an intracellular receptor from the nuclear receptor superfamily termed the AR. To confirm the presence of microglial AR and to assess the APOE isoform-specific differences in AR expression, mRNA levels for AR in microglia derived from APOE3 mice were compared with microglia from APOE4 mice using real time PCR. AR mRNA levels in APOE3 microglia were not significantly different from levels expressed by APOE4 microglia (data not shown). Protein levels, as determined by Western blot, were not significantly different (data not shown). We also examined AR mRNA and protein expression in cortical brain samples of adult male APOE3 and APOE4 mice (n = 3 male mice per genotype). No significant difference was observed in AR, estrogen receptor {alpha}, or estrogen receptor ß (data not shown).

Because the action of androgens, like other steroid hormones, can be mediated by classical genomic and by nongenomic signaling pathways (41, 42, 43, 44, 45), we investigated changes in the activity of specific signaling pathways known to be involved in the inflammatory effects of androgens. For these experiments, primary microglial cultures from APOE3 and APOE4 mice were treated for 30 min with LPS + IFN{gamma} alone or in the continuing presence of DHT after pretreatment (15 h) with varying concentrations of DHT. Phosphorylated and nonphosphorylated forms of p38 MAPK (Fig. 4AGo), JNK p54/p46 (Fig. 4BGo), and ERK1/2 (Fig. 4CGo) were detected by Western blot using specific antibodies. In each case, phosphorylation was increased when microglia were treated with LPS + IFN{gamma}, confirming previously published findings on kinase activity in immune-activated macrophages (41). APOE genotype was shown to significantly affect p38 and JNK46/54 phosphorylation in immune-activated microglia treated with DHT (Fig. 4Go, D and E). A significant interaction was observed for both phospho-p38 (P < 0.008; F(3,16) = 5.6) and phospho-JNKp54/p46 (P < 0.003; F(3,16) = 7.34). In contrast, ERK1/2 phosphorylation was not affected by either genotype or treatment with DHT (Fig. 4FGo).


Figure 4
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  		FIG. 4. APOE genotype-specific differences in phosphorylation of MAPK pathway components by DHT. Microglia from APOE3 and APOE4 mice were pretreated with varying doses of DHT (0–10 nM) and then immune activated with LPS + IFN{gamma} for 30 min to determine the levels of phosphorylated and nonphosphorylated MAPK. Representative Western blots are shown for p38 (A), JNK p54/p46 (B), and ERK1/2 MAPK (C) pathways. Upper panels for each enzyme represent the phosphorylated form as detected by specific anti-phospho-antibodies, whereas lower panels represent total p38, JNKp54/p46, and ERK1/2 using appropriate specific antibodies detecting total enzyme level. The relative density of phospho-p38 (D), phospho-JNK p54 (E), and phospho-ERK1 (F) compared with total levels of each kinase are also shown. Significant interactions between APOE genotype and DHT treatment were observed for p-p38 (P < 0.008) and p-JNKp54 (P = 0.0026) but not for p-ERK. *, P < 0.05, **, P < 0.01, ***, P < 0.001 for DHT treated compared with LPS + IFN{gamma} alone (0 DHT); #, P < 0.05; ###, P < 0.001 for APOE4 compared with APOE3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DHT treatment of primary microglia derived from neonatal mice expressing the human APOE3 gene reduced LPS + IFN{gamma}-stimulated NO and TNF{alpha} production in a dose-dependent fashion. These data confirm the antiinflammatory effect of androgens reported in the literature and shows that DHT directly modulates macrophage function (46, 47, 48, 49). In contrast, treatment of microglia with testosterone produce increased levels of nitrite consistent with a proinflammatory effect. The apparently opposite effects of DHT and testosterone on the microglial immune response cannot be simply explained by the conversion of testosterone to estrogens by aromatase. DHT is a highly specific agonist for AR and DHT cannot be converted to estrogen by aromatase (50). However, under some conditions, macrophages may express aromatase (51) and 5{alpha}-reductase (52), and thus testosterone can undergo aromatization to 17ß-estradiol or reduction to DHT, respectively. As a result, testosterone’s action may reflect a more complex pattern of outcomes depending on the steroidogenic enzymes present within macrophages. Although cultured microglia express mRNA for estrogen receptors {alpha} and ß, they do not express aromatase mRNA under untreated conditions or after LPS stimulation (data not shown). In addition, 17ß-estradiol has been shown to act as a direct antiinflammatory agent for microglia (35, 53). As a result, testosterone is unlikely to generate its proinflammatory effects in microglia through an estrogenic mechanism.

Peritoneal macrophages derived from adult APOE3 mice also demonstrate a complex response to testosterone and both proinflammatory and antiinflammatory effects are observed. The exact reasons for these differences in testosterone’s actions are unclear but may reflect additional modulatory events produced by age of the animal or by prior exposure to circulating steroid hormones including androgens.

Our data on APOE3 microglia and peritoneal macrophages confirm and extend the role of androgens in immune regulation. However, our study uniquely focuses on androgen-mediated immune regulation in mice expressing only the human APOE3 gene compared with mice expressing only the human APOE4 gene. These data clearly demonstrate that the antiinflammatory activity of DHT is suppressed in APOE4 microglia. A statistically significant interaction between APOE genotype and the response to DHT was found for nitrite and cytokine production in immune-activated microglia. Interestingly, the genotype effect was specific for DHT treatment because the testosterone-mediated regulation of immune-activated microglia was similar for both APOE3 and APOE4 microglia. The reasons for the opposing effect of APOE genotype on testosterone and DHT-mediated immune regulation are unclear but may reflect different inherent mechanisms of action of DHT and testosterone on microglia.

Our data also suggest that the effect of APOE4 on macrophage function is not limited to CNS microglia and extends to peripheral macrophages derived from adult mice. Replacement of endogenous testosterone in GDX male APOE3 mice increased immune-activated nitrite production and decreased TNF{alpha} production. The opposing results of testosterone are puzzling, but NO, itself, may contribute to TNF{alpha} regulation. NO has been shown to both up and down-regulate TNF{alpha} in macrophages through biphasic regulation of nuclear factor {kappa}B (NF{kappa}B) (54). Regardless, the response to testosterone in APOE4 macrophages was significantly different from the response in APOE3 macrophages.

To understand the failure of APOE4 microglia to respond to androgens in the same manner as APOE3 microglia, we measured the expression levels of AR mRNA and protein. Previous studies using immunocytochemistry have shown that microglia within the CNS express AR (24, 55). Studies using more indirect functional measures in the presence of specific AR antagonists have further confirmed the presence of ARs. Our data show that microglia in culture express AR, but there is no difference in expression levels between APOE3 and APOE4 cultures. Expression levels for AR in cortex from APOE3 and APOE4 mice are also not significantly different. Raber et al. (34), using a binding assay to determine AR levels, demonstrated decreased binding in the neocortex of APOE4 mice compared with APOE3 mice, but no isoform-specific change in binding in the hippocampus. However, this data cannot be directly compared with our current findings because those studies used mice wherein APOE expression is under control of the neuron-specific promoter, neuron-specific enolase. In contrast, the APOE targeted replacement mice used in our study express the human apolipoprotein E isoform under the normal mouse APOE gene’s promoter and thus, allow a more normal physiological response to be detected.

To further examine isoform-specific differences in the mechanism of action of androgens on immune-activated microglia, we measured kinase activation in untreated microglia and in microglia treated with LPS + IFN{gamma} alone or in the presence of physiological doses of DHT (1–10 nM). A specific pattern of intracellular signaling has been well described for LPS + IFN{gamma} immune-activated macrophages and invariantly includes activation of the MAPK pathways (37, 56, 57). As a result, rapid phosphorylation of p38 MAPK, JNK p54/p46, and ERK1/2 occurs and is followed by activation of NF{kappa}B and the transcription of proinflammatory target genes. IFN{gamma} serves as a "priming" agent for this response, in part, by up-regulating the expression of critical pathway components (58). Our data clearly demonstrate that LPS + IFN{gamma}-stimulated phosphorylation of p38 MAPK and JNK p54/p46 but not ERK1/2 are inhibited by DHT in APOE3 microglia but are not inhibited to the same degree in APOE4 microglia. Testosterone treatment of a clonal macrophage cell line, RAW cells, has been reported to also inhibit p38 MAPK in association with inhibition of NO production (28, 41). However, Guo et al. (41) did not find changes in JNK p54/p46 or ERK1/2 phosphorylation and also found that cyproterone, an anti-androgen that inhibits intracellular AR action, did not block the inhibition of p38 MAPK phosphorylation. The multiple differences between our study and Guo et al. (41) make it difficult to compare these data directly. However, the strong similarities between these two studies imply that nonclassical actions of androgens are a prominent feature of macrophagic cells including primary microglia. Importantly, the signaling pathway between DHT and kinase activation is altered in the APOE4 microglia. Compared with APOE3 microglia, DHT treatment did not block the phosphorylation of p38 MAPK or JNK p54/p56 in APOE4 microglia. Our data suggest that signaling pathway defects may underlie these APOE genotype-specific differences. However, the exact cellular site of this defect remains unclear and may reflect a dysfunction in the Toll 4-like receptor that responds to LPS or other steps of the inflammatory cascade of intracellular signaling pathways.

The inability of APOE4 microglia and peripheral macrophages to respond in a normal manner to androgen is likely to produce a change in the profile of the innate immune response in the CNS and in the periphery. APOE4 microglia and macrophages are less sensitive to androgens and thus are associated with prolonged or constitutive activation of the MAPK and NF{kappa}B pathways to extend rather than limit inflammation. This potential mechanism of action is consistent with the observation that increased markers of inflammation are observed in association with the APOE4 genotype (35, 36, 59, 60, 61).

In summary, our results show that androgens modulate the inflammatory response in a complex manner. However, the presence of the APOE4 gene significantly decreases that response and demonstrates that genotypic factors can impact androgen-mediated regulatory events. Because of the widespread importance of the APOE gene to numerous inflammatory processes, including CNS dysfunction and atherosclerosis, the change in androgen activity is likely to have human health consequences. At the least, the underlying mechanisms illuminated by our results strongly suggest that simple replacement of physiological androgen levels in males and females expressing an APOE4 genotype may not be an adequate therapy for limiting inflammatory disease processes.


   Acknowledgments
 
We thank Dr. H. Barnhard (Biostatistics and Bioinformatics, Duke University Medical Center) for her help with the manuscript.


   Footnotes
 
This work was supported by a grants to C.A.C. from the National Institutes of Health (AG023802-01), to M.P.V. (AG019780), and C.M.B. (Minority Research Supplement to P50 AG05128) and an National Science Foundation Predoctoral Fellowship (to C.M.B.).

Present address for C.M.B.: Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195.

Present address for N.O.: Department of Physiology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon-city, Ehime 791-0295, Japan.

First Published Online March 29, 2007

Abbreviations: AD, Alzheimer’s disease; APOE, apolipoprotein A; AR, androgen receptor; CNS, central nervous system; DHT, dihydrotestosterone; FBS, fetal bovine serum; GDX, gonadectomy; IFN{gamma}, interferon-{gamma}; JNK, Janus kinase; LPS, lipopolysaccharide; NF{kappa}B, nuclear factor {kappa}B; NO, nitric oxide.

Received August 31, 2006.

Accepted for publication March 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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