Estrogen protects the blood–brain barrier from inflammation-induced disruption and increased lymphocyte trafficking
Introduction
Several lines of evidence suggest that the sex differences seen in vascular and neural diseases may be at least partly linked to differing sex hormone complements. In particular, a number of beneficial effects have been attributed to the principal female hormone estrogen, including in conditions as diverse as Parkinson’s disease, Alzheimer’s disease, head injury and multiple sclerosis (Gillies and McArthur, 2010). One important working hypothesis is that the neuroprotective effects of estrogen may be related to its known anti-inflammatory and immunomodulatory actions (Czlonkowska et al., 2005, Nadkarni and McArthur, 2013).
The endothelium of the blood–brain barrier (BBB) is at the forefront of the defensive features of the central nervous system, regulating its interactions with the immune system. In particular, there is accumulating evidence that BBB function is compromised during peripheral inflammation, leading to inappropriate passage of cells and molecules into the brain parenchyma (Carvey et al., 2009). Estrogen has been shown to exert a variety of anti-inflammatory effects, including reducing iNOS activity (Cignarella et al., 2009), directly regulating the cytokine milieu (Gameiro et al., 2010) and altering expression of vascular and leukocyte adhesion molecules (Dietrich, 2004). Whilst initial studies have identified estrogen as being able to modulate BBB tight junction proteins such as claudin 5 (Burek et al., 2014), full characterization of the effects of estrogen upon the BBB is lacking, and represents an underexplored aspect of hormonal protection.
We have previously studied the importance of the anti-inflammatory protein annexin A1 (ANXA1) in the BBB, where it plays a major role in the regulation of tight junction expression, contributing to limited barrier permeability (Cristante et al., 2013). Although ANXA1 was originally described as a glucocorticoid second messenger (Flower and Blackwell, 1979), we and others have since reported that it can also be modulated by estrogen (Solito et al., 2003, Davies et al., 2007, Nadkarni et al., 2011), leading us to hypothesize that estrogen may exert protective effects upon the BBB in inflammation through the regulation of this protein.
Using a combined in vivo/in vitro approach, we examined the response of the BBB to peripheral inflammatory challenge and the ability of the principal estrogen: estradiol (E2) to restore homeostasis in this system. We report here the presence of sexual dimorphism in the response of the BBB to peripheral pro-inflammatory challenge in vivo, and a dual protective role for estradiol upon the inflamed cerebral endothelium in vitro, mediated through the regulation of ANXA1 and ICAM-1 expression. Together these actions control movement of both small molecules and immune cells across the cerebral endothelium.
Section snippets
Reagents
All reagents are from Sigma–Aldrich (Poole, UK) unless otherwise stated. Cell culture medium and solutions were purchased from Lonza (Basel, Switzerland). The ERα agonist PPT (4,4′,4″-(4-propyl-(5)-pyrazole-1,3,5-triyl)trisphenol), the ERβ agonist DPN (2,3-bis(4-Hydroxyphenyl)-propionitrile), the GPR30 agonist G-1 ((±)-1-[(3aR∗,4S∗,9bS∗)-4-(6-bromo-1,-3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone), the ERβ antagonist PHTPP
Estrogen-dependent sex differences in the BBB response to inflammatory challenge
We initially characterized the response of the murine BBB to a simple systemic inflammatory model, the administration of bacterial lipopolysaccharide (LPS; 3 mg/kg i.p.), with assessment of barrier permeability through extravasation of intravenous Evan’s blue dye in young adult (2 month) male and female C57Bl/6 mice. A marked sex difference was notable in male animals showing a clear and reproducible, but transient, increase in BBB permeability 4 h post-LPS administration, whilst such change was
Discussion
Steroid hormones have long been known to possess anti-inflammatory properties, indeed synthetic glucocorticoid receptor agonists are amongst the most clinically potent anti-inflammatory drugs available, but it is only relatively recently that the anti-inflammatory potential of estrogen has begun to be closely studied (Nadkarni and McArthur, 2013). The primary interface between the CNS and the inflammatory response, the BBB, has been examined as a target for estrogen action (Oztas et al., 2001,
Conclusions
The prevalence of inflammatory cerebrovascular diseases shows a distinct male predominance, with numerous studies showing women to be relatively protected. Understanding why this is the case remains a challenge. Here, we identify a powerful protective action of the female sex steroid estrogen, showing that this hormone can both enhance inter-endothelial cell tight junction function and limit lymphocyte extravasation following challenge with inflammatory cytokines. Moreover, we identify these
Author contributions
Elisa Maggioli: Performed in vivo and in vitro experiments, data analysis.
Simon McArthur: Performed in vivo work, the immunocytochemical and confocal microscopic analysis, qPCR, data analysis, contributed to writing and discussion of the manuscript.
Claudio Mauro: Performed shRNA lentivirus production and contributed to discussion of the manuscript.
Julius Kieswich: Animal handling and support in vivo experiments.
Dennis Kusters: Performed ANXA1 expression and purification.
Chris Reutelingsperger:
Financial interest
The authors declare no conflict of interests.
Acknowledgments
This work was supported by ARUK – PPG2013B-2 and FISM-3/15/F14 to ES, Barts and the London Trust to MY, and British Heart Foundation Fellowship FS/12/38/29640 to CM.
References (48)
- et al.
Structure and function of the blood–brain barrier
Neurobiol. Dis.
(2010) - et al.
Mechanisms of transcriptional activation of the mouse claudin-5 promoter by estrogen receptor alpha and beta
Mol. Cell. Endocrinol.
(2014) Mouse syngenic in vitro blood–brain barrier model: a new tool to examine inflammatory events in cerebral endothelium
Lab. Invest.
(2005)- et al.
Menopause and aging: changes in the immune system—a review
Maturitas
(2010) - et al.
Systemic immune activation shapes stroke outcome
Mol. Cell. Neurosci.
(2013) - et al.
Oestrogen and immunomodulation: new mechanisms that impact on peripheral and central immunity
Curr. Opin. Pharmacol.
(2013) - et al.
Sex-dependent changes in blood–brain barrier permeability in epileptic rats following acute hyperosmotic exposure
Pharmacol. Res.
(2001) - et al.
Neuroprotective effects of estrogens following ischemic stroke
Front. Neuroendocrinol.
(2009) Linking thrombophilia and idiopathic intracranial hypertension
J. Lab. Clin. Med.
(2005)Anti-allergic drugs and the Annexin-A1 system
Pharmacol. Rep.
(2010)
Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood–brain barrier
J. Cell Sci.
Stroke increases g protein-coupled estrogen receptor expression in the brain of male but not female mice
Neurosignals
Claudin-5 as a novel estrogen target in vascular endothelium
Arterioscler. Thromb. Vasc. Biol.
The blood–brain barrier in neurodegenerative disease: a rhetorical perspective
J. Neurochem.
Distinct roles of estrogen receptor-alpha and beta in the modulation of vascular inducible nitric-oxide synthase in diabetes
J. Pharmacol. Exp. Ther.
Feature Article: Identification of an essential endogenous regulator of blood–brain barrier integrity, and its pathological and therapeutic implications
Proc. Natl. Acad. Sci. U.S.A.
Estrogen and cytokines production – the possible cause of gender differences in neurological diseases
Curr. Pharm. Des.
The influence of 17beta-estradiol on annexin 1 expression in the anterior pituitary of the female rat and in a folliculo-stellate cell line
J. Endocrinol.
Endothelial cells of the blood–brain barrier: a target for glucocorticoids and estrogens?
Front. Biosci.
Anti-inflammatory steroids induce biosynthesis of a phospholipase A2 inhibitor which prevents prostaglandin generation
Nature
Lymphocytes: potential mediators of postischemic injury and neuroprotection
Stroke
Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines
Pharmacol. Rev.
Lactate regulates metabolic and pro-inflammatory circuits in control of t cell migration and effector functions
PLoS Biol.
Sex differences in stroke
J. Cereb. Blood Flow Metab.
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EM and SMcA contributed equally to this work.