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. 2010 Aug 4;151(10):4916–4925. doi: 10.1210/en.2010-0371

Production of Proinflammatory Cytokines and Chemokines During Neuroinflammation: Novel Roles for Estrogen Receptors α and β

Candice M Brown 1, Tara A Mulcahey 1, Nicole C Filipek 1, Phyllis M Wise 1
PMCID: PMC2946152  PMID: 20685874

Abstract

Neuroinflammation is a common feature of many neurological disorders, and it is often accompanied by the release of proinflammatory cytokines and chemokines. Estradiol-17β (E2) exhibits antiinflammatory properties, including the suppression of proinflammatory cytokines, in the central nervous system. However, the mechanisms employed by E2 and the role(s) of estrogen receptors (ERs) ERα and ERβ are unclear. To investigate these mechanisms, we employed an in vivo lipopolysaccharide (LPS) model of systemic inflammation in ovariectomized (OVX) and OVX and E2-treated (OVX+E2) mice. Brain levels of proinflammatory cytokines (IL-1β, IL-6, and IL-12p40) and chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, and CXCL1/KC) were quantified in mice at 0 (sham), 3, 6, 12, and 24 h after infection using multiplex protein analysis. E2 treatment inhibited LPS-induced increases in all cytokines. In contrast, E2 treatment only suppressed CCL/RANTES chemokine concentrations. To determine whether ERα and ERβ regulate brain cytokine and chemokine levels, parallel experiments were conducted using ERα knockout and ERβ knockout mice. Our results revealed that both ERα and ERβ regulated proinflammatory cytokine and chemokine production through E2-dependent and E2-independent mechanisms. To assess whether breakdown of the blood-brain barrier is an additional target of E2 against LPS-induced neuroinflammation, we measured Evan’s blue extravasation and identified distinct roles for ERα and ERβ. Taken together, these studies identify a dramatic cytokine- and chemokine-mediated neuroinflammatory response that is regulated through ERα- and ERβ-mediated ligand-dependent and ligand-independent mechanisms.

Estrogen receptors α and/or β are necessary for appropriate regulation of brain proinflammatory cytokines and chemokines and for blood-brain barrier permeability.

A complex and dynamic series of interactions in the immune, endocrine, and central nervous systems (CNS) governs the brain’s innate immune response. Under normal physiological conditions, the immune cells of the brain engage in continual immune surveillance to ensure that the neuronal environment is protected from both central and peripheral insults. During pathological states, such as brain injury, neurodegenerative disease, and septic shock, a systemic inflammatory response is initiated, and bidirectional communication mechanisms convey information between the brain and periphery. Maintaining a precise balance between proinflammatory and antiinflammatory immune signals is therefore critical for an appropriate innate immune response (1,2).

Sex steroids strongly influence neuroimmune communication pathways. A strong sexual dimorphism exists as part of the immune response, and estrogens are responsible, in part, for many sex differences (3,4). Furthermore, clear sex differences have emerged in neurological disorders, whose disease mechanisms are accompanied by chronic inflammation, including Alzheimer’s disease, Parkinson’s disease, stroke, and multiple sclerosis (5). Estradiol-17β (E2) is the most potent endogenously synthesized and secreted ovarian estrogen. It is a pleiotropic hormone with reproductive, as well as nonreproductive, roles in the nervous system, cardiovascular system, immune system, adipose tissue, and bone (5).

E2 mediates many of its biological effects through the classical estrogen receptors (ERs), ERα and ERβ. ERs participate in both classical and nonclassical signaling pathways. Classical pathways regulate transcription through direct interactions with an estrogen response element (ERE). In contrast, nonclassical pathways employ indirect, ERE-independent regulation of transcription through other cis-acting promoter elements (e.g. nuclear factor κB, activator protein-1, and cAMP response element-binding protein) or through membrane-bound ER of activation of second messenger pathways that induce transcription using ERE-dependent or ERE-independent mechanisms (6). Both ERs are expressed throughout the CNS in microglia, astrocytes, and neurons. ERs are also expressed in many types of immune cells, including monocytes/macrophages, T cells, B cells, dendritic cells, and natural killer cells (4). Both ERα and ERβ play a role in mediating the antiinflammatory properties of E2 in the CNS and in the immune system (4,7).

Peripheral stimuli, such as lipopolysaccharide (LPS) evoke significant cytokine responses in the CNS that are an integral part of the brain’s innate inflammatory response (1,8). Physiological and pharmacological concentrations of E2 exhibit potent antiinflammatory activity in the CNS by suppressing production of proinflammatory cytokines, such as IL-6, IL-1β, and TNFα in many neurological disorders (reviewed in Ref. 9). Cytokines are secreted proteins that convey autocrine or paracrine information among cells of the immune system. Apart from studies of multiple sclerosis using the experimental autoimmune encephalomyelitis (EAE) animal model, it is less clear whether physiological concentrations of E2 also suppress brain proinflammatory chemokines as well (10). Chemokines, or chemotactic cytokines, are classically known as regulators of leukocyte infiltration and separated into two primary groups based on structural motifs, which, in turn, confer functional specificity. The CC chemokines, or β-chemokines, contain a conserved cysteine-cysteine motif, whereas the CXC chemokines, or α-chemokines, all share a conserved motif containing two cysteine residues separated by a single amino acid (11). Although several studies have demonstrated a critical role for ERα in mediating the antiinflammatory activity of E2 in neurological disease models (12,13,14), the role of ERβ and the mechanisms employed by both ERα and ERβ to regulate proinflammatory cytokines and chemokines are not well understood.

The loss of ovarian estrogens in postmenopausal women may exacerbate central and peripheral inflammatory responses that occur with normal aging. One of the most prominent changes in the brain’s neuroinflammatory response during aging is the increased and/or poorly regulated production of proinflammatory mediators, a phenomenon referred to as “inflamm-aging” (15). A growing body of evidence suggests that this dysfunctional response is further exacerbated by the decline in ovarian estrogens precipitated by the menopause. Blood levels of proinflammatory cytokines, such IL-6 and TNFα, increase significantly in postmenopausal women compared with their premenopausal counterparts (16,17,18,19), thereby contributing to a chronic, subclinical state of inflammation that may contribute to neurodegeneration and cognitive decline (15,20). In young and middle-aged rodents subjected to ovariectomy, a model that mimics surgical menopause, proinflammatory cytokine production increases in several injury models in the both the CNS and the periphery, whereas treatment with physiological levels of E2 attenuates increases in cytokine production (12,21).

There is increasing interest in understanding the mechanisms of communication between the CNS and the periphery that coordinate a unique immune response to protect the brain from peripheral as well as neurological insult. The blood-brain barrier (BBB), a component of the neurovascular unit, orchestrates the movement of cytokines and other signals from the periphery to the brain and also conveys information to the periphery from the brain’s immune system (22). E2 regulates the selective permeability of the BBB in several models of injury, including systemic inflammation and ischemia (reviewed by Ref. 23). The mechanisms employed by E2 and the role(s) of its receptors, ERα and ERβ, in regulating BBB permeability are also unclear.

Many studies that address the antiinflammatory properties of E2 in the brain and periphery have yielded different results depending on many factors, including the disease model, species, experimental outcome, whether the study is in vivo or in vitro, estrogenic formulation, and concentration of E2 or other estrogens (4,5). To further elucidate the antiinflammatory mechanisms employed in vivo by E2 in the CNS, we addressed the hypothesis that low, physiological levels of E2 suppress brain proinflammatory cytokines and chemokines through its receptors, ERα and/or ERβ. Using a comprehensive and systematic approach, we used our in vivo model to assess: 1) which cytokines and chemokines are induced in the brain after peripheral LPS stimulation, 2) whether these molecules are suppressed by low, physiological levels of E2, 3) how E2 impacts BBB permeability, and 4) whether the classical ERs, ERα and ERβ, exhibit distinct or complementary roles as regulators of the brain’s inflammatory response by extending our experimental paradigm to ERα null and ERβ null mice. Our findings reveal novel roles for ERα and ERβ as mediators of cytokine and chemokine regulation in the CNS.

Materials and Methods

Animals and surgical procedures

Animal studies were performed in compliance with National Institutes of Health guidelines and were approved by the University of Washington Institutional Animal Care and Use Committee. All mice used in this study were housed in University of Washington vivaria. ERαKO (24) and ERβKO (25) breeding pairs (n = 3) were generously provided by Dr. Kenneth Korach (National Institute of Environmental Health Sciences). Both strains were backcrossed at least eight generations to C57BL/6J mice. Mice were genotyped for both wild-type (WT) and KO alleles using RedTaq PCR mix (Sigma, St. Louis, MO) along with the following primer sets and cycling conditions in an ABI 9700 thermocycler (Applied Biosystems, Foster City, CA). ERαKO mice were genotyped using the following primer sets: 1) IMR013, 5′-CTTGGGTGGAGAGGCTATTC-3′; IMR014, 5′-AGGTGAGATGACAGGAGATC-3′, and 2) IMR3100, 5′-CGGTCTACGGCCAGTCGGGGCACC-3′; IMR3101, 5′-GTAGAAGGCGGGAGGGCCGGTGTC-3′. These primer sets were used in two separate reactions for the presence of the KO and WT bands, respectively, and amplified under the following conditions: 95 C, 5 min hot start, followed by 35 cycles: 94 C for 30 sec, 64 C for 1 min, 72 C for 90 sec, and 72 C for 2 min extension. Primers IMR013 and IMR014 amplified a 280-bp KO band indicative of the presence of the neomycin resistance gene; primers IMR3100 and IMR3101 amplified a 239-bp WT band. ERβKO mice were genotyped using the following primers: 6122, 5′-GCAGCCTCTGTTCCACATACAC-3′; C1-2, 5′-CATCCTTCACAGGACCAGACAC-3′; and mERβF2, 5′-TGGACTCACCACGTAGGCTC-3′ in a single multiplexed reaction and amplified under the following conditions: 95 C for 5 min hot start, followed by 35 cycles: 95 C for 30 sec, 57 C for 60 sec, 72 C for 60 sec, and 72 C for 7 min extension in a single reaction. These primers produce a 404-bp KO band and/or a 356-bp WT band. All WT (C57BL/6J) mice were either purchased from The Jackson Laboratory–West (Sacramento, CA) or were littermates of ERαKO or ERβKO mice.

All surgical procedures were performed under specific pathogen-free conditions. WT, ERαKO, and ERβKO female mice (10–15 wk of age) were anesthetized using isoflurane, bilaterally ovariectomized (OVX), and treated with SILASTIC (Dow Corning Corp., Midland, MI) capsules containing sesame oil (Sigma) vehicle (OVX+oil) or E2 (OVX+E2, 180 μg/ml; Sigma) as previously described (26). E2 capsules produce serum levels of E2 (25–30 pg/ml) equivalent to low physiological levels in intact females with regular estrous cycles (27,28).

LPS systemic injection and tissue collection

Seven days after OVX, all mice (excluding sham animals) received a single, sublethal injection of LPS (Escherichia coli 055:B5 5 mg/kg, ip; Sigma) based on previously published paradigms shown to induce a robust cytokine response in the brain (29,30); all LPS stocks were prepared in sterile, pyrogen-free saline. Brain tissue was collected at 3, 6, 12, or 24 h after LPS injection; a sham (control) group was also collected for each genotype. At the termination of the experiment, mice were deeply anesthetized with ketamine/xylazine (130 mg/kg/8.8 mg/kg). Brains were extracted and sliced into 1-mm coronal sections with razor blades using a chilled 1-mm stainless steel brain matrix; 1-mm brain sections were subsequently mounted onto ice-cold slides, rapidly frozen on dry ice, and stored at −80 C for further analysis. Uteri were visually inspected for signs of atrophy in appropriate experimental groups.

Brain lysate preparation

A 1-mm brain section within the following region (bregma 1.32 mm, interaural 5.12 mm to bregma 0.0 mm, and interaural 3.3 mm) was selected from each animal and used for further analysis. This brain region encompasses a section of cortex and striatum. All brain tissues were homogenized using sonication in Tissue Extraction Reagent I (Invitrogen, Camarillo, CA) supplemented with Complete Mini Protease Inhibitor Tablets (Roche, Indianapolis, IN) according to manufacturer’s instructions. Brain lysate protein concentrations were determined using the Pierce BCA Protein Assay kit (Pierce Biotechnology, Rockford IL) and absorbance measured at 562 nm using a Tecan Infinite M200 microplate reader (Tecan, Männedorf, Switzerland).

Cytokine/chemokine quantification

Assays to determine cytokine and chemokine concentrations in brain tissue were performed by Assaygate (Ijamsville, MD) using a multiplex bead-based suspension assay as previously described by our laboratory with minor modifications (21). This methodology is similar to the Luminex bead-based immunoassay platform. All samples were assayed at 1 mg/ml in duplicate. The limit of detection (LOD) for each analyte was determined by calculating the concentration on the standard curve lying two sds above the mean background median fluorescence intensity for ten replicates. The LOD for all analytes was as follows: IL-1β, 7 pg/ml; IL-6, 2 pg/ml; IL-12p40, 2 pg/ml; interferon-γ (IFNγ), 6 pg/ml; TNFα, 6 pg/ml; CXCL1/keratinocyte chemoattractant/KC, 3 pg/ml; CCL2/monocyte chemoattractant protein-1 (MCP-1),14 pg/ml; CCL3/macrophage inflammatory protein-1α (MIP-1α), 24 pg/ml; and CCL5/regulated upon activation, normal T-cell expressed, and secreted (RANTES), 5 pg/ml. Analyte concentrations below the threshold of detection were excluded from the analysis. For the remainder of this paper, we will use the standard chemokine nomenclature and refer to molecules with CC and CXC designations (31).

Evan’s blue (EB) extravasation

In a separate set of experiments, WT, ERαKO, and ERβKO mice were analyzed for BBB permeability using EB dye (Sigma) using modifications of previously published methods (32,33,34). Two hours before the termination of the experiment, mice received tail vein injections of 2% EB (7.5 μl/g body weight). At the end of experiment, mice were deeply anesthetized as described above and transcardially perfused with 10–15 ml of 0.1 m PBS (pH 7.0), followed by dissection of the liver and brain, excluding olfactory bulbs, hypothalamus, and cerebellum. Samples were weighed and homogenized via sonication in 2 ml (brain) or 3 ml (liver) 50% trichloroacetic acid, followed by overnight incubation in the dark at room temperature to complete the extraction of EB from all tissues. After centrifugation of samples at 17,000 × g for 30 min, the supernatant was diluted 1:3 with 95% ethanol and fluorescence measured using a Tecan microplate reader (excitation, 620 nm; emission, 680 nm). EB concentrations were determined using a standard curve prepared in 50% trichloroacetic acid/95% ethanol (1:3). Final EB concentrations were expressed as ng EB/mg wet weight tissue. Brain EB concentrations were normalized to EB liver concentrations from the same mouse to control for differences in the total amount of EB actually injected and expressed as a ratio.

Statistical analysis

All data are expressed as mean ± sem with P < 0.05 as significant. Data were analyzed using two-way ANOVA with Bonferroni’s post hoc comparisons using GraphPad Prism 5.0 (GraphPad, San Diego, CA).

Results

We used ip LPS injection, an in vivo model of systemic inflammation, to assess the contribution of E2 and the classical ERs, ERα and ERβ, as regulators of the cytokine and chemokines in the brain. After OVX and treatment with vehicle or low physiological levels of E2, we measured brain concentrations of proinflammatory cytokines and chemokines in a region spanning the cortex and striatum. Cytokine and chemokine levels were below the LOD at 0 h (sham) in all experiments. IFNγ and TNFα were undetectable at all time points monitored in this study (data not shown).

E2 suppresses brain proinflammatory cytokines

To determine whether E2 suppressed proinflammatory cytokines, we measured concentrations of IL-1β, IL-6, and IL-12p40 from WT brain lysate at 0 (sham), 3, 6, 12, and 24 h after acute LPS challenge. Figure 1 shows a dramatic elevation of all cytokines in OVX mice, which was attenuated by E2 treatment: IL-1β (P < 0.02) (Fig. 1A), IL-6 (P < 0.05) (Fig. 1B), and IL-12p40 (P < 0.0001) (Fig. 1C). Two-way ANOVA also revealed a significant interaction between time and hormone treatment for IL-12p40 (P < 0.0001).

Figure 1.

Figure 1

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Brain proinflammatory cytokines are suppressed by E2 after a peripheral LPS stimulus in WT mice. Mice were injected with LPS (ip), and brain levels of cytokines were measured using a multiplex cytokine assay in sham (0 h) animals and at 3, 6, 12, and 24 h after LPS injection. Low physiological levels of E2 significantly decreased production of (A) IL-1β [interaction between time and hormone (F(1,51) = 3.11, P < 0.03), time (F(4,51) = 14.69, P < 0.0001), and hormone (F(1,51) = 6.57, P < 0.02)]; (B) IL-6 [time (F(4,50) = 21.61, P < 0.0001) and hormone (F(1,50) = 4.13, P < 0.05)]; and (C) IL-12p40 [interaction between time and hormone (F(4,43) = 9.49, P < 0.0001), time (F(4,43) = 44.07, P < 0.0001), and hormone (F(1,43) = 21.51, P < 0.0001)]. *, P < 0.05 and ***, P < 0.0001 indicates significance between oil and E2; n = 5–9 mice/hormone group/time point.

E2 signaling regulates brain proinflammatory cytokine levels through ERα and ERβ

The next set of studies addressed whether ERα and/or ERβ mediated the antiinflammatory properties of E2 for the production of IL-1β, IL-6, and IL-12p40 in the brain. Based on data from WT mice, we selected three groups, i.e. 0 (sham), 6, and 24 h, to address our hypothesis in ERαKO and ERβKO mice using the same treatment paradigm. Because we did not measure any significant differences in cytokine production for sham animals in WT, ERαKO, or ERβKO mice (data not shown), we focused our analysis on cytokine production at 6 and 24 h after LPS injection. Figure 2 shows that the antiinflammatory properties of E2 are eliminated in ERαKO and ERβKO mice at 6 h for IL-1β (P < 0.05) (Fig. 2A) and IL-6 (P < 0.05) (Fig. 2C). In contrast, the antiinflammatory properties of E2 were lost in ERαKO but retained in ERβKO mice for IL-12p40 (P < 0.01) (Fig. 2E), thus demonstrating a critical role for E2-dependent activation of ERα. Post hoc analysis showed that E2 treatment significantly decreased IL-12p40 concentrations in WT and ERβKO mice compared with ERαKO mice (P < 0.05). At 24 h, levels of IL-1β (Fig. 2B), IL-6 (Fig. 2D), and IL-12p40 (Fig. 2F) did not differ significantly between WT, ERαKO, or ERβKO mice.

Figure 2.

Figure 2

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ERα and ERβ mediate the antiinflammatory effects of E2 on proinflammatory cytokine production. ERαKO and ERβKO mice were injected with LPS (ip) and brain levels of cytokines measured using a multiplex cytokine assay at 6 h (A, C, and E) and 24 h (B, D, and F) after LPS injection. A, IL-1β production was higher in both ERαKO and ERβKO mice compared with WT mice [F(2,26) = 24.01, P < 0.03]. C, IL-6 production was also significantly higher in ERαKO and ERβKO mice compared with WT mice [F(2,29) = 20.36, P < 0.03]. E, IL-12p40 production was suppressed by E2 treatment [F(2,28) = 23.7, P < 0.003] in WT and ERβKO mice; this effect was abolished in ERαKO mice. There were no significant differences at 24 h for (B) IL-1β, (D) IL-6, and (F) IL-12p40. *, P < 0.05 indicates significance between oil and E2; #, P < 0.05 indicates significance between WT and ERαKO or ERβKO; ERαKO, n = 4–7/time point; ERβKO, n = 5–8/time point.

Chemokines are induced in the brain after peripheral LPS injury

Because we identified a role for E2 as a regulator of proinflammatory cytokine production in the brain, our next set of experiments addressed whether E2 also regulates proinflammatory chemokines in the brain after peripheral LPS challenge. Proinflammatory chemokines were selected based on prior studies that showed they were either induced after LPS challenge and/or they were regulated by E2 (4,8,17). Figure 3 shows that the chemokine CCL5 was induced over time (P < 0.0001) (Fig. 3A) and was significantly elevated in OVX mice compared with E2-treated counterparts at 12 h (P < 0.01) and 24 h (P < 0.05) after injury. In contrast, CCL2, CCL3, and CXCL1, were all induced in the brain after peripheral LPS challenge, but this increase was not abated by E2 treatment. Two-way ANOVA revealed a main effect of time but no effect of E2 for CCL2 (P < 0.0001) (Fig. 3B), CCL3 (P < 0.0001) (Fig. 3C), and CXCL1 (P < 0.0001) (Fig. 3D). In contrast, CCL5 was induced over time (P < 0.0001) (Fig. 3A) and was significantly elevated in OVX mice compared with E2-treated counterparts at 12 h (P < 0.01) and 24 h (P < 0.05) after injury.

Figure 3.

Figure 3

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Peripheral LPS induces brain proinflammatory chemokines. Mice were injected with LPS (ip) and brain levels of cytokines measured using a multiplex cytokine assay in sham (0 h) animals and at 3, 6, 12, and 24 h after LPS-injection. A, CCL5 [F(4,49) = 29.17, P < 0.0001] was induced by LPS and suppressed by E2 [hormone (F(1,49) = 12.28, P < 0.002)]; B, CCL2 [F(4,52) = 28.4, P < 0.0001]; C, CCL3 [F(4,46) = 12.08, P < 0.0001]; and D, CXCL1 [F(4,51) = 29.87, P < 0.0001] were induced by LPS but unaffected by E2. *, P < 0.05 indicates significance between oil and E2; n = 5–9 mice/hormone group/time point.

ERα and ERβ modulate brain chemokine levels

Because we found that E2 suppressed CCL5 production in the brain at the later stages (12 h and 24 h) of acute LPS infection, we assessed whether ERα or ERβ play a role in regulating this response using ERαKO and ERβKO mice. There was a significant effect of genotype in both ERαKO and ERβKO mice, because CCL5 concentrations were significantly suppressed at 12 h (P < 0.001) (Fig. 4A) and 24 h (P < 0.001) (Fig. 4B) in OVX+oil ERαKO and ERβKO mice compared with WT mice. In contrast, CCL5 concentrations were unchanged in OVX+E2-treated WT and ERKO mice. This finding suggests a ligand-independent role for ERα and ERβ in the regulation of CCL5 levels during neuroinflammation. Because previous studies have suggested that ERα and ERβ can regulate the expression of some genes in a ligand-independent manner, we next assessed whether CCL2, CCL3, or CXCL1 production was altered in ERαKO and ERβKO mice. In contrast, chemokine concentrations were significantly increased at 24 h in ERαKO and ERβKO mice from both OVX+oil and OVX+E2-treated groups for CCL2 (P < 0.001) (Fig. 4C), CCL3 (P < 0.001) (Fig. 4D), and CXCL1 (P < 0.001) (Fig. 4E). Chemokine concentrations were also measured in sham (0 h) animals after LPS injection, and there were no significant changes in sham ERKO mice compared with sham WT mice (data not shown).

Figure 4.

Figure 4

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Brain chemokine induction is regulated by both ERα and ERβ. ERαKO and ERβKO mice were injected with LPS (ip), and brain levels of chemokines were measured using a multiplex cytokine assay after LPS-injection. CCL5 levels were measured at 12 (A) and 24 h (B) after injury. A significant interaction between ER genotype and hormone was observed at 12 h [F(2,23) = 10.83, P < 0.001], as well as an effect of genotype [F(2,23) = 10.24, P < 0.001] and hormone [F(1,23) = 7.973, P < 0.01]. The interaction between ER genotype and hormone persisted at 24 h [F(2,25) = 3.958, P < 0.04], in addition to an effect of genotype [F(2,25) = 3.533, P < 0.05]. In contrast, chemokine production was significantly higher after 24 h in both ERαKO and ERβKO mice as measured by a significant effect of ER genotype for (C) CCL2 [F(2,33) = 11.12, P < 0.001], (D) CCL3 [F(2,27) = 12.16, P < 0.001], and (E) CXCL1 [F(2,26) = 11.46, P < 0.001]. *, P < 0.05 indicates significance between oil and E2; #, P < 0.05 and **, P < 0.01 indicates significance between WT and ERαKO or ERβKO; ERαKO, n = 4–7/time point; ERβKO, n = 5–8/time point.

ERα and ERβ are essential for E2-mediated regulation of BBB permeability

In our final set of experiments, we addressed whether E2 modulates levels of proinflammatory cytokines and chemokines by altering the permeability of the BBB after peripheral LPS challenge. WT mice were subjected to LPS challenge as in prior experiments and were injected with 2% EB 2 h before the termination of the experiment in sham animals (0 h), as well as at 6 and 24 h after LPS challenge. Quantification of EB in mouse brain tissue showed that BBB permeability is significantly increased over the 24-h time course of LPS challenge in both OVX+oil and OVX+E2-treated WT mice (P < 0.01) (Fig. 5A). Post hoc analysis revealed significant differences in BBB permeability between OVX+oil and OVX+E2-treated mice at 6 h (P < 0.05) (Fig. 5A).

Figure 5.

Figure 5

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ERα mediates E2-dependent suppression of BBB permeability. A, EB permeability is decreased in OVX+E2-treated WT mice at 6 h after LPS injection [F(2,33) = 5.627, P < 0.01]; n = 5–8. B, E2-mediated suppression of EB permeability is lost in ERαKO mice and retained in ERβKO mice as shown by a significant interaction between genotype and hormone [F(2,29) = 3.763, P < 0.05] and an effect of genotype [F(2,29) = 18.9, P < 0.001)]. *, P < 0.05 indicates significance between oil and E2; #, P < 0.05 and ###, P < 0.0001 indicates significance between WT and ERαKO or ERβKO; ERαKO and ERβKO, n = 5–7.

Because we detected significant hormone differences between OVX +oil and OVX+E2-treated mice at 6 h, we next tested whether BBB permeability was differentially regulated in ERαKO or ERβKO mice 6 h after LPS injection. We identified a significant interaction between hormone treatment and genotype as regulators of BBB permeability (P < 0.05) (Fig. 5B). In addition, we identified an effect of ER genotype (P < 0.0001), because overall BBB permeability significantly increased in OVX+oil -and OVX+ E2-treated ERαKO mice. However, the hormone difference observed in WT mice also persisted in ERαKO mice. Conversely, BBB permeability was not significantly different in ERβKO mice compared with WT mice, but the suppressive effect of E2 was abolished in ERβ deficient mice. Post hoc analysis revealed that BBB permeability significantly decreased in OVX+oil and OVX+E2-treated ERβ KO mice compared with their ERαKO counterparts (P < 0.05) (Fig. 5B).

Discussion

Our study employed an in vivo approach to investigate the roles of E2, ERα, and ERβ as regulators of CNS cytokine and chemokine production over the time course of an acute, peripheral inflammatory stimulus. The results reveal several novel findings that contribute to our understanding of the role(s) of E2 and the classical ERs, ERα and ERβ, as regulators of the brain’s innate inflammatory response. First, we used a multiplex approach to measure multiple cytokines and chemokines and showed that peripheral LPS challenge induces changes in multiple proinflammatory cytokine (IL-1β, IL-6, and IL-12p40) and chemokine (CCL2, CCL3, CCL5, and CXCL1) levels in the brain. Second, we show that low, physiological levels of E2 differentially regulate brain cytokines and chemokines after LPS injury. Third, using ERαKO and ERβKO mice, we demonstrate that both receptors participate in E2 dependent and E2 independent regulation of cytokine and chemokine production during neuroinflammation. Finally, we show that ERα, but not ERβ, is required for the E2-mediated suppression of BBB permeability. Taken together, our findings reveal novel E2-dependent and E2-independent roles for ERα and ERβ in neuroinflammation.

These results demonstrate that a cascade of proinflammatory cytokines and chemokines are induced in the brain after injury. Our results confirm previous findings that E2 suppresses brain IL-1β and IL-6 (21,35) after injury and extend them to include IL-12p40. Regardless of E2 treatment, both IL-1β and IL-6 levels were increased in ER-deficient mice after LPS injury. This suggests that both cytokines are regulated by ligand-dependent and ligand-independent ER mechanisms in the brain. Our results also extend previous findings showing that ERα mediates the antiinflammatory activity of E2 in the brain to include ERβ. ERβ’s antiinflammatory activity has been demonstrated in microglia (36) and astrocytes (37). We also identified a novel role for ERα as a regulator of the proinflammatory properties of IL-12p40 in the brain. IL-12p40 also plays a critical role in the innate immune response, and it serves primarily as a macrophage chemoattractant during injury. IL-12p40 gene expression is induced in microglia after LPS treatment in vivo and in vitro (38,39), and we extend these findings to define an antiinflammatory role for ERα in mediating the proinflammatory properties of IL-12p40 in the brain.

Less is known about the regulation of chemokines by sex steroids in the brain. With the exception of EAE, few in vivo studies have addressed whether E2 and/or ERs regulate brain chemokines (10). Many chemokines also act as neuromodulators and are constitutively present in the brain at low, basal levels under normal physiological conditions, in addition to being induced after injury (40). Recent studies have uncovered novel roles for chemokines as neuromodulators with varied roles in brain development, neuronal survival and repair, neurogenesis, behavior, and neuroendocrine function (11,40). We identified a novel, E2 independent role for ERs as regulators of CCL5, a chemokine whose functions in normal physiology and pathophysiology are not well understood. In contrast to our results for other chemokines, we found that genetic ablation of ERα and ERβ decreased rather than increased brain CCL5 levels. These results suggest that CCL5 production and activity is tightly regulated in the brain and that CCL5 might exhibit both proinflammatory and neuromodulatory roles after injury. Recent studies have identified heterogeneous roles for CCL5 as a regulator of neuronal survival (41,42) and as a suppressor of neurotoxicity in endothelial cells (42) and microglia (43). We also report the novel finding that ERα and ERβ regulate CXCL1, a chemokine that recruits neurotrophils into the CNS and peripheral tissues after injury. Previous work has shown that peripheral injection of LPS promotes neutrophil recruitment via induction of CXCL1 across the brain parenchyma (44). Our results suggest that ERα and ERβ are both necessary for appropriate regulation of neutrophil recruitment into the brain. In contrast to previous studies in models of EAE, stroke and primary macrophage culture, E2 did not attenuate CCL2 or CCL3 levels in our LPS-induced model of neuroinflammation (10,21,45,46,47). However, this result is not surprising, because systemic LPS challenge, EAE, and middle cerebral artery occlusion are disease models that exhibit diverse signaling mechanisms.

Although many studies have reported a primary role for ERα as a regulator of the antiinflammatory properties of E2, a growing number of studies has reported antiinflammatory roles for ERβ as well. Treatment with ERβ agonists suppresses transcription of IL-6, TNFα, and CCL2 (48), decreases IL-1β levels in astrocytes and microglia (37), and improves survival in mouse models of sepsis (49). Our results reveal novel ligand-dependent and ligand-independent roles for ERα and ERβ as regulators of the magnitude of cytokine and chemokine production in the brain. The molecular mechanisms employed by ERs to regulate cytokine and chemokine production are complex and not clearly understood. Many cytokine and chemokine gene promoters lack a classical ERE. In addition, ERs also exhibit ligand-independent activation mechanisms. Using the ERE-luciferase reporter mouse, Ciana et al. (50) reported ERE-dependent transcriptional activity in adult OVX mice. Furthermore, both in vitro and in vivo studies show that ERs can be activated in the absence of ovarian hormones by coregulators, growth factors, or by ER phosphorylation (51). Our results from ERαKO and ERβ KO ovariectomized mice suggest that ERα and ERβ participate in pathways involving ligand-independent activation of ERs to regulate chemokine production in the brain. Finally, a growing body of work also supports a pivotal role for cross talk of classical and nonclassical signaling mechanisms for ER signaling in multiple tissues, including the brain (52).

Our finding that OVX increases BBB permeability is consistent with other studies performed with both mice (53) and rats (33,34,54,55). Furthermore, our results using ER null mice demonstrate that ERα is required for E2-mediated regulation of BBB permeability. Endothelial tight junctions between cells promote BBB integrity and E2 has been shown to regulate several tight junctions proteins (56,57,58). Although both ERα and ERβ are expressed on endothelial cells, ERα appears to play a critical role in mediating the actions of E2 on endothelial cells (59,60). Due to the complexities of ER signaling pathways, it is difficult to identify the precise events underlying E2- and ER-mediated mechanisms at the BBB (2). Based on the design of our study, it is unclear whether cytokines and chemokines are produced within the CNS or enter the CNS from the periphery through compromised regions of the BBB, via receptors located on the BBB, or are secreted by endothelial cells on the BBB (23,61). Several cytokines measured in this study are secreted from choroid plexus ependymal cells and endothelial cells of the BBB, including IL-1, IL-6, CCL2, and CCL5 (23). Furthermore, cytokines and chemokines are also produced in the brain primarily by microglia and astrocytes, with an additional contribution from both neurons and endothelial cells (11,40). Previous in vivo (12,62) and in vitro (37,63) studies have shown that E2 attenuates the LPS-induced activation of microglia and astrocytes.

Use of an in vivo approach in ERαKO and ERβKO mouse models may have affected the interpretation of our results. It is possible that direct or indirect regulatory mechanisms could have developed in either or both ERKO mouse models and contributed to our findings. Future studies using ERα- and/or ERβ-specific agonists and antagonists will allow us to gain additional insights by combining genetic approaches with complementary pharmacological approaches. Another limitation of our model is that we measured brain levels of cytokines and chemokines rather than the production of these molecules from individual CNS cell types or from infiltrating peripheral immune cells that cross the BBB. Nevertheless, our genetic model yielded novel insights on the induction patterns of specific cytokines and chemokines after systemic endotoxemia, the role of ERs as regulators of neuroinflammation, and the contribution of the BBB to this mechanism.

There are abundant sex differences in both acute and chronic inflammatory conditions encompassing sepsis, cardiovascular disease, and neurodegenerative disease. An emerging hypothesis to explain the greater susceptibility of postmenopausal women compared with premenopausal women to a worse outcome in acute and chronic inflammatory pathologies posits that the postmenopausal period is associated with low-grade systemic inflammation that extends to the CNS (5,16,19). Injury and disease exacerbate low-grade systemic inflammation by providing a second hit, which, ultimately, results in greater proinflammatory responses, including the increased production of cytokines and chemokines that lead to a more severe course of disease. Taken together, this study suggests that targeted therapeutics, such as ERα and/or ERβ selective ER modulators, could potentially reduce neuroinflammatory responses associated with acute systemic infections and extend these treatments to women who suffer from neurodegenerative disease. In summary, our results shed important insights on the role of E2 and its classical receptors, ERα and ERβ, as regulators of cytokine, and more importantly, chemokine responses in the brain. Our emerging understanding of the role(s) of chemokines and their receptors in the brain, and how E2 might influence their functions as both proinflammatory mediators and neuromodulators, makes them exciting therapeutic targets for further study.

Footnotes

This work was supported by National Institutes of Health grants AG002224 (to P.M.W.) and F32AG027614 (to C.M.B.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online August 4, 2010

Abbreviations: BBB, Blood-brain barrier; CCL, CC ligand; CXCL, CXC ligand; CNS, central nervous system; E2, estradiol-17β; EAE, experimental autoimmune encephalomyelitis; EB, Evan’s blue; ER, estrogen receptor; ERE, response element; ERαKO, ERα knockout; ERβKO, ERβ knockout; IFNγ, interferon-γ; LOD, limit of detection; LPS, lipopolysaccharide; OVX, ovariectomized; WT, wild type.

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