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1521-0081/70/2/278–314$35.00
https://doi.org/10.1124/pr.117.014647PHARMACOLOGICAL REVIEWS
Pharmacol Rev 70:278–314, April 2018Copyright © 2018 by The
Author(s)This is an open access article distributed under the CC
BY-NC Attribution 4.0 International license.
ASSOCIATE EDITOR: ROBERT DANTZER
Neuroimmune Axes of the Blood–Brain Barriers andBlood–Brain
Interfaces: Bases for PhysiologicalRegulation, Disease States, and
Pharmacological
InterventionsMichelle A. Erickson and William A. Banks
Geriatric Research and Education and Clinical Center, Veterans
Affairs Puget Sound Health Care System, Seattle, Washington;
andDivision of Gerontology and Geriatric Medicine, Department of
Medicine, University of Washington, Seattle, Washington
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . 279I. Introduction. .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . 280
A. The Blood–Brain Barrier and Immune Privilege . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
280B. Working Definitions of Brain Barriers and Interfaces . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
280C. Historical Work: Defining the Brain Barriers . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . 280
II. Features and Functions of the Blood–Brain Barrier and
Blood–Brain Interface . . . . . . . . . . . . . . . 281A.
Specialized Features That Confer Barrier Functions . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
1. Tight Junctions . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . 2812. Reduced Macropinocytosis. . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . 2823. Efflux Transporters .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2824.
Metabolic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . 282
B. Specialized Features That Confer Interface Functions . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2821. Transcellular Diffusion. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . 2822. Blood–Brain Barrier Transport via Solute
Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . 2833. Blood–Brain Barrier Transport via
Receptor-Mediated Transcytosis . . . . . . . . . . . . . . . . . .
. 2834. Blood–Brain Barrier Transport via Adsorptive Transcytosis .
. . . . . . . . . . . . . . . . . . . . . . . . . . 283
C. From Brain Barriers to Brain Interfaces: Components of the
Neurovascular Unit . . . . . . . . . 2841. Endothelial Cells. . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2842.
Brain Pericytes . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . 2843. Astrocytes . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 2854. Neurons .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . 2865. Microglia and Perivascular Macrophages . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . 2866. Mast Cells . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . 2867. Extracellular
Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2868. Glycocalyx . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . 287
D. Arms of the Blood–Brain Barrier and Their Neuroimmune
Functions. . . . . . . . . . . . . . . . . . . . . 2871. The
Vascular Blood–Brain Barrier . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2872. The Blood–Cerebrospinal Fluid Barrier . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2873. Tanycytic/Ependymal Barriers of Circumventricular Organs .
. . . . . . . . . . . . . . . . . . . . . . . . . 288
III. The Neuroimmune Axes . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . 289A. Axis 1: Blood–Brain Barrier
Disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . 289
1. Disruption of Paracellular Tight Junctions. . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2902. Transcytotic Vesicular Pathways . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . 2903. Endothelial Cell Damage and Hemorrhage . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . 291
B. Axis 2: Modulation of Barrier and Interface Functions by
Immune Substances. . . . . . . . . . . . 291
Address correspondence to: Dr. William A. Banks, Veterans
Affairs Puget Sound Health Care System, Room 810A/Building 1, 1660
S.Columbian Way, Seattle, WA 98108. E-mail: [email protected]
This work was supported by the Department of Veterans Affairs
and the National Institutes of Health National Institute on Aging
[GrantR01 AG046619].
https://doi.org/10.1124/pr.117.014647.
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C. Axis 3: Transport, Penetration, and Uptake of
Neuroimmune-Related Substances. . . . . . . . . 292D. Axis 4:
Immune Cell Trafficking between Blood and Brain . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . 294E. Axis 5: Immune
Secretions of the Barrier Cells . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . 296
IV. Physiologic Conditions, Disease States, and Pharmacologic
Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . 297A.
Sickness Behavior . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . 297B. Perinatal Brain Ischemia . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . 297C. Multiple
Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . 298D. Human Immunodeficiency Virus-1 Penetration of
the Blood–Brain Barrier and
Consequences of Human Immunodeficiency Virus-1 Infection on
Blood–Brain BarrierFunction . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 299
E. Amyloid b Peptide and Alzheimer Disease . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . 300F. Neuromyelitis Optica . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . 301G. Euthyroid Sick Syndrome . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 301H.
Chemobrain . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . 301I. Inflammation, ATP-Binding
Cassette Transporters, and Central Nervous System Drug
Delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . 302J. Emergence of
Progressive Multifocal Leukoencephalopathy Associated with
Therapies
That Inhibit T-Cell Immune Surveillance . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . 303K. IL-1ra, Febrile Infection-Related Epilepsy Syndrome, and
Neonatal-Onset Multisys-
tem Inflammatory Disease. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . 303L. Immunomodulatory Therapies and Stroke . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . 304M. Methamphetamine and Blood–Brain Barrier
Alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . 304N. Cerebral Cavernous Malformations . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . 304O. Antibody-Associated Autoimmune
Encephalitis Syndromes . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . 304
V. Conclusions and Future Directions. . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . 305Acknowledgments . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
306References . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . 306
Abstract——Central nervous system (CNS) barriers pre-dominantly
mediate the immune-privileged status of thebrain, and are also
important regulators of neuroimmunecommunication. It is
increasingly appreciated thatcommunication between the brain and
immune systemcontributes to physiologic processes, adaptive
responses,and disease states. In this review, we discuss the
highlyspecialized features of brain barriers that
regulateneuroimmune communication in health and disease. Insection
I, we discuss the concept of immune
privilege,provideworkingdefinitionsofbrainbarriers,
andoutlinethehistoricalwork that contributed to
theunderstandingofCNS barrier functions. In section II, we discuss
the uniqueanatomic, cellular, and molecular characteristics of
the
vascular blood–brain barrier (BBB), blood–cerebrospinalfluid
barrier, and tanycytic barriers that confer theirfunctions as
neuroimmune interfaces. In section III, weconsider BBB-mediated
neuroimmune functions andinteractions categorized as five
neuroimmune axes:disruption, responses to immune stimuli, uptake
andtransport of immunoactive substances, immune celltrafficking,
and secretions of immunoactive substances.In section IV, we discuss
neuroimmune functions ofCNS barriers in physiologic and disease
states, as wellas pharmacological interventions for CNS
diseases.Throughout this review, we highlight many recent
advancesthat have contributed to the modern understanding ofCNS
barriers and their interface functions.
ABBREVIATIONS: Ab, amyloid b; ABC, ATP-binding cassette; AD,
Alzheimer disease; AJ, adherens junction; ART, anti-retroviral
therapy; BALT,bronchus-associated lymphoid tissue; BBB, blood–brain
barrier; BCRP, breast cancer resistance protein; BCSFB, blood–CSF
barrier; BEC, brainendothelial cell; BUI, brain uptake index; CCL,
C-C motif chemokine ligand; CICD, chemotherapy-induced cognitive
dysfunction; CMB, cerebralmicrobleed; CNS, central nervous system;
COX, cyclooxygenase; CP, choroid plexus; CPE, choroid plexus
epithelium; CSF, cerebrospinal fluid; CVO,circumventricular organ;
DHA, docosahexaenoic acid; DOX, doxorubicin; EAE, experimental
autoimmune encephalomyelitis; ECM, extracellular matrix;FIRES,
febrile infection-related epilepsy syndrome; GRP78,
glucose-regulated protein 78; HAART, highly active antiretroviral
therapy; HAND, HIV-associated neurologic disorder; HIV, human
immunodeficiency virus; HIVE, HIV encephalitis; IDT, indicator
diffusion technique; IFN, interferon; IL,interleukin; IL-1R1, type
1 IL-1 receptor; ISF, interstitial fluid; JAM, junctional
adhesionmolecule; LPC, lysophosphatidylcholine; LPS,
lipopolysaccharide;LRP, low-density lipoprotein receptor-related
protein; LTP, long-term potentiation; ME, median eminence; MFSD2A,
major facilitator superfamilydomain-containing 2A; MMP, matrix
metalloproteinase; MRP, multidrug resistance protein; MS, multiple
sclerosis; NF-kB, nuclear factor kB; NLR,nucleotide-binding
oligomerization domain-like receptor; NMO, neuromyelitis optica;
NNRTI, non-NRTI; NOD, nucleotide-binding oligomerizationdomain;
NRTI, nucleoside reverse transcriptase inhibitor; NVU,
neurovascular unit; OVLT, organum vasculosum of the lamina
terminalis; PDGF-B,platelet-derived growth factor subunit B;
PDGFRb, platelet-derived growth factor receptor b; Pgp,
P-glycoprotein; Plvap, plasmalemma vesicle-associatedprotein; PML,
progressive multifocal leukoencephalopathy; RAGE, receptor for
advanced glycation endproducts; RMT, receptor-mediated
transcytosis; T3,triiodothyronine; TEER, transendothelial
electrical resistance; TGF, transforming growth factor; TJ, tight
junction; TNF, tumor necrosis factor; TSH,thyroid-stimulating
hormone; VCAM, vascular cell adhesion molecule; VEGF, vascular
endothelial growth factor; ZO, zonula occulins.
BBB and Neuroimmune Axes 279
-
I. Introduction
A. The Blood–Brain Barrier and Immune Privilege
The central nervous system (CNS) has traditionallybeen viewed as
an immune-privileged area in that it isprotected against the immune
events of the periphery.This immune privilege was once considered
absolutewith its violation only occurring in disease
states,resulting in dire consequences for the CNS.
Currentappreciation is that the immune privilege is real,
butrelative. Both past and current thinking ascribes thisimmune
privilege to the BBB. By its ability to prevent(past thinking) or
to control and modulate (currentthinking) the impact of peripheral
immune events onthe CNS, the BBB acts to protect the brain
fromperipheral immune events. However, whereas thebarrier aspects
of the BBB induce immune privilege, it isalso the BBB that makes
such protection relative. TheBBBmakes immune privilege relative by
possessing waysin which it controls the interplay of CNS and
peripheralimmune events. Such interplay relies on the transfer
ofimmune elements (substances or cells) between the CNSand blood;
such a transfer can be termed a neuroimmuneaxis. Five neuroimmune
axes can be currently identifiedthat involve the BBB and a sixth
that does not but ismediated by afferent and efferent nerve
activities (Goehleret al., 1999; Romeo et al., 2001; Kelley et al.,
2003; Kenneyand Ganta, 2014; Kanashiro et al., 2016). The
fivepathways involving the BBB are as follows: 1) BBBdisruption;
2)modulation of barrier and interface functions(other than BBB
integrity) by immune substances; 3)transport, penetration, and
uptake of neuroimmune-related substances; 4) immune cell
trafficking betweenblood and brain; and 5) immune secretions of the
barriercells. Most likely, these axes work together rather
thanindependently, and it is one of the great promises of thefield,
as well as one of its greatest challenges, that it willelucidate
the mechanisms of neuroimmune integrationthat underlie such diverse
phenomena as sleep, responsesto sepsis, mindfulness, and
depression. Below, we considereach of the five axes involving the
BBB.This review will first consider the concepts, compo-
nents, functions, and interactions that form the basis ofthe BBB
field that are germane to neuroimmunology,then discuss how the
BBBdefines and participates in thefive known neuroimmune axes, and
finally discuss howthe BBB in general and how these axes in
particular areinvolved in CNS diseases, drug delivery, and
therapy.
B. Working Definitions of Brain Barriersand Interfaces
BBB is a term that refers to one function of a highlyspecialized
cellular interface between the blood and theCNS parenchyma. The
barrier function of this interfaceprevents unregulated diffusion of
circulating sub-stances into the brain, which is critical for
maintaininga CNS milieu that supports neuronal function and
survival. Generally, the BBB refers to the vascularbarrier where
capillary endothelial cells are the in-terface between the blood
and brain parenchyma.Specialized vascular CNS barriers that have
distinctfeatures from brain parenchymal vessels include
theblood–retinal barrier, the blood–nerve barrier,
theblood–labyrinth barriers, and the blood–spinal cordbarrier
(Neuwelt et al., 2008). Other cellular barriers/interfaces such as
the choroid plexus epithelial cells ofthe blood–cerebrospinal fluid
(CSF) barrier (BCSFB)and tanycytes that are located along the
ventricularboundaries of circumventricular organs (CVOs) may
beconsidered arms of the BBB as well because they alsoprevent
unregulated leakage of blood components intothe CSF and adjacent
brain interstitial fluid (ISF)(Ghersi-Egea et al., 1996).
Furthermore, these inter-faces have many nonbarrier functions that
are essentialin supporting CNS homeostasis. The nonbarrier
func-tions include regulating the transport of
circulatingsubstances into the brain, removing potentially
harmfulsubstances from the brain, secreting molecules thatsignal to
cells in the brain parenchyma, and respondingto stimuli that arise
within both the brain and bloodcompartments. In the remainder of
this section, we willprovide an overview of the historical work
that definedthe barrier functions of brain interfaces,
discussaspects of their barrier and interface functions
thatcontribute to CNS homeostasis, and relate eachblood–brain
interface to one another with regard totheir unique structures and
functions in neuroim-mune communication.
C. Historical Work: Defining the Brain Barriers
Evidence in support of a BBB dates back to workspublished in the
late 19th/early 20th century. At thetime, the field of medicinal
chemistry was in its infancy,and Paul Ehrlich, who would later make
many seminalcontributions to the field (Bosch and Rosich, 2008),
wasinvestigating the selectivity of dyes for different cellsand
tissues. As part of this work, Ehrlich (1885) notedthat certain
water-soluble dyes that were parenterallyinjected into animals
stained peripheral tissues and thechoroid plexus, but did not stain
the brain or spinal cordparenchyma. Ehrlich (1906) posited that the
reducedCNS staining was due to a low affinity of the dyes forCNS
tissue. However, other groups who were investi-gating bile acids
(Biedl and Kraus, 1898) and sodiumferrocyanide (Lewandowski, 1900)
found that the toxic-ities of these substances were much more
potent whenintroduced directly into the cerebrospinal fluid
versussystemically. Both groups interpreted their findings tomean
that brain capillaries had unique properties thatblocked the
transfer of certain molecules from blood tobrain. Goldmann (1909,
1913) later conducted criticalexperiments showing that Ehrlich’s
trypan blue dye,which did not bind CNS tissue when injected
parenter-ally, did stain CNS tissue when injected into the CSF.
280 Erickson and Banks
-
This finding demonstrated that exclusion of trypan bluefrom the
CNS was not due to reduced binding affinity toCNS tissue, which had
been suggested by Ehrlich(1906). However, Goldmann (1913) supposed
in thiswork that the choroid plexus was the predominantbarrier site
of the CNS, supplying nutrients to theCNS in a fashion analogous to
the placenta. The termBBB (barrière hémato-encéphalique) was first
used in apublication by Stern andGautier (1921);
althoughmanyattribute the first use of this term
(Blut-Hirnschranke)to Lewandowski, this term was not used in his
originalpublication (Saunders et al., 2014).Following these early
studies, controversy sur-
rounded the true nature of the BBB. Early ultrastruc-tural
studies of the brain using electron microscopyused methods of
tissue preservation that led to thebelief that the CNS had
essentially no extracellularspace (Bradbury, 2000). Therefore, some
adopted thebelief that the limited CNS penetration of
aqueoussolutes was due to tightly packed neuronal and
glialmembranes and a lack of aqueous medium for diffusion(Davson
and Spaziani, 1959; Bradbury, 2000). However,it was then
demonstrated that extracellular markersinjected in ventricular CSF
did penetrate the brain andspinal cord, and therefore, CNS tissue
contained extra-cellular fluid permissive to solute diffusion
(Davson andSegal, 1969). Based on these findings, it was
positedthat a BBB to such solutes injected in blood must
exist(Davson et al., 1961). Later that decade, Vanharreveldet al.
(1965) showed that the brain extracellular spacecould be visualized
by electron microscopy when mod-ified techniques were used for
tissue preservation. Thisreport was followed by the eminent
findings of Reeseand Karnovsky (1967), who used electron microscopy
toexplore the subcellular features of brain capillaries.They found
that the brain’s limited uptake of peroxi-dase, which had been
shown previously (Straus, 1958),could be attributed to two
specialized features of thebrain endothelium: uniquely impermeant
tight junc-tions (TJs) that were present at contacts
betweencapillary membranes, and markedly reduced endothe-lial
vesicles (Reese and Karnovsky, 1967). In a laterstudy, Brightman
and Reese (1969) explored the distri-bution of peroxidase injected
into CSF of mice, chickens,and goldfish to determine which cell
types of the CNSexpressed peroxidase-impermeant TJs. Although
per-oxidase did diffuse through gap junctions that werepresent at
contacts of astrocytic endfeet, it did notpermeate the junctions
between brain endothelial cells(BECs) or choroid plexus ependymal
cells. This workhighlighted that TJs were a unique feature of BECs,
aswell as epithelial cells of the choroid plexus thatconferred
barrier properties (Brightman and Reese,1969). The tight barriers
of the brain vasculature werefurther exemplified by Crone and
Olesen (1982) and byButt et al. (1990), who showed that brain
vessels haveexceptionally low ion permeabilities due to very
high
transendothelial electrical resistance (TEER), averagingover
1000 V/cm2 in frog and mammalian pial vessels. Aspial vessels lack
astrocytes, TEER is estimated to bemuch higher in brain parenchymal
vessels that areensheathed by astrocyte endfeet, which contribute
tothe BBB phenotype (Abbott et al., 2006). By determiningthe CNS
permeability/surface area coefficients for K, Na,and Cl in vivo,
Smith and Rapoport (1986) estimatedTEER of the parenchymal vessels
to be approximately8000 V/cm2, which compares to that of a cell
membrane.
II. Features and Functions of the Blood–BrainBarrier and
Blood–Brain Interface
A. Specialized Features That ConferBarrier Functions
Barrier functions are largely thought of as thoserelated to the
exclusion of blood-borne substances fromthe brain. Such exclusion
is critical to the CNS being animmune-privileged tissue. The
specialized features thatunderlie those functions include TJs,
mechanisms thatlimit macropinocytosis, efflux mechanisms, and
enzy-matic activities.
1. Tight Junctions. TJs are macromolecular com-plexes that
prevent the paracellular diffusion of solutesacross brain barriers.
They communicate with adherensjunctions (AJs), which are another
junctional complexthat contributes to the tightness of the barrier
(Wolburgand Lippoldt, 2002). AJs are located at the basalmembrane
and are comprised of cadherins, whichassociate with each other in
the extracellular space,and catenins, which link the cadherins to
the cytoskel-eton (Huber et al., 2001). TJ proteins at the
vascularBBB include occludin, claudins, junctional
adhesionmolecules (JAMs), zonula occludens (ZO), and cytoplas-mic
accessory proteins such as cingulin (Hawkins andDavis, 2005).
Occludins, claudins, and JAMs aremembrane-spanning proteins that
heterotypically in-teract in the intercellular space between
adjacentcapillary membranes. ZOs and cingulin are adaptorproteins
that link TJ membrane proteins to the cyto-skeleton and contribute
to junction stability (Abbottet al., 2010). TJ stability is
regulated by calcium,phosphorylation, cyclic AMP, and G proteins
(Huberet al., 2001), and decreases in TJ expression as well asTJ
mislocalization and post-translational modificationscan cause
increases in paracellular permeability ofsolutes across the BBB
(Deli et al., 2005). Importantly,TJs are not only a diffusion
barrier for transcellularpassage of circulating substances into the
CNS, but alsofor the lateral diffusion of membrane proteins
(Abbottet al., 2010). Therefore, TJs also contribute to themembrane
polarity of BECs. Later in this review, wewill compare TJ
organization in the different arms ofthe BBB as well as the
contributions of TJs to theneuroimmune axes of the BBB.
BBB and Neuroimmune Axes 281
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2. Reduced Macropinocytosis. Reese and Karnovsky(1967) were the
first to note that BECs had relativelylow numbers of vesicles
compared with endothelial cellsin the periphery. In contrast,
epithelial cells of thechoroid plexus do have vesicles that largely
reside attheir apical membranes (Johanson et al., 2011). Recentwork
has begun to identify the molecular mechanismsthat suppress brain
endothelial pinocytic vesicle forma-tion. In a study that aimed to
characterize gestationaldevelopment of the BBB, Ben-Zvi et al.
(2014) foundthat the BBB tightening that occurred around E15.5
inmice was associated with upregulation of major facili-tator
superfamily domain-containing 2A (MFSD2A) inBECs. In the same
study, it was shown that MFSD2Aknockout mice had a leaky BBB,
although the morphol-ogy of the brain vasculature and TJs did not
appear tobe affected. Instead, MFSD2A knockout mice had in-creased
luminal, abluminal, and cytoplasmic vesicles intheir BECs, which
facilitated the fluid-phase uptake ofsolutes such as peroxidase and
dextrans, which areusually excluded from the CNS. In a parallel
study, itwas realized thatMFSD2A knockoutmice had cognitiveand
behavioral symptoms that resembled omega-3fatty-acid deficiency
(Nguyen et al., 2014). Using alipidomics approach, this group
revealed that docosa-hexaenoic acid (DHA), an omega-3 fatty acid
that isimportant for CNS development and cognition, wasreduced in
the CNS of mice lacking MFSD2A. Theyfurther demonstrated that
MFSD2A was a transporterfor lysophosphatidylcholine (LPC)-DHA, as
well asLPC-oleate and LPC-palmitate at slightly lower capac-ities
(Nguyen et al., 2014). Andreone et al. (2017) thenshowed that the
DHA-transporting function ofMFSD2A also facilitated DHA enrichment
of BECmembranes, which inhibited caveolin-1–induced forma-tion of
vesicles. In mice lacking MFSD2A, caveolin-1knockout inhibited the
increased formation of brainendothelial vesicles as well as leakage
of the BBB(Andreone et al., 2017). Therefore, BECs acquire
aspecialized lipid composition during embryonic devel-opment that
inhibits caveolae-mediated fluid-phasetranscytosis.3. Efflux
Transporters. Efflux transporters at brain
barriers facilitate the passage of substances in
thebrain-to-blood or CSF-to-blood direction. A subset ofefflux
transporters, namely members of the ATP-binding cassette (ABC)
protein family, confers barrierfunctions by limiting the brain
uptake of endogenousmacromolecules and xenobiotics. ABC transporter
sub-types, their localization, and functions in the CNS havebeen
extensively reviewed elsewhere (Hartz and Bauer,2011). Some of the
most studied ABC transporters atthe BBB include P-glycoprotein
(Pgp/ABCB1), multi-drug resistance protein (MRPs/ABCC), and
breastcancer resistance protein (BCRP/ABCG2) (Qosa et al.,2015).
ABC transporters have a broad substrate spec-trum, including some
phospholipids, sphingolipids,
aldosterone, and amyloid b (Ab) for Pgp;
glutathione,glutathione-conjugated leukotrienes and
prostaglandins,and glucoronidation and sulfation products for
MRP-1;and bile acids and estrones for BCRP (Qosa et al., 2015).ABC
transporters also efflux a broad range of xenobioticsubstrates,
including opioids, antibiotics, antiretroviraldrugs,
chemotherapeutics, and others (Qosa et al., 2015).The expression
and function of efflux transporters at theBBB are dynamically
regulated, for example, at thetranscriptional level by nuclear
receptors (Chan et al.,2013), and at post-translational levels by
vascular endo-thelial growth factor (VEGF) (Hawkins et al.,
2010).Aspects of ABC transporter modulation
regardingimmune-regulated functions are discussed in greaterdetail
later in this review.
4. Metabolic Enzymes. The BBB is also an enzy-matic barrier, and
expresses phase I and phase IIenzymes that contribute to the
metabolism and elimi-nation of biomolecules and drugs from the
body. BECscontain monoamine oxidase and
catechol-O-methyltransferase, which metabolize adrenaline,
nor-adrenaline, and dopamine, as well as
4-aminobutyrateaminotransferase, which metabolizes GABA
(Lasbenneset al., 1983; Spatz et al., 1986). Therefore,
enzymaticbarriers inhibit the transport of many neurotransmit-ters
in the blood-to-brain and brain-to-blood directions.Furthermore,
the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine when
systemically administered isinversely associated with monoamine
oxidase expres-sion in BECs (Kalaria et al., 1987; Riachi and
Harik,1988). The cytochrome P450 enzyme CYP1B1 isexpressed in human
BECs (Dauchy et al., 2008;Shawahna et al., 2011), and its
expression can beregulated by environmental toxicants such
as2,3,7,8-tetrachlorodibenzo-p-dioxin through the arylhydrocarbon
receptor (Jacob et al., 2015). GlutathioneS-transferases are also
expressed in human braincapillaries (Shawahna et al., 2011).
B. Specialized Features That ConferInterface Functions
Brain barriers do much more than divide the CNSfrom the
peripheral circulation. They are also critical inCNS homeostasis,
nutrition, and brain–body communi-cation. These features are
essential to the existence ofsome of the neuroimmune axes. Features
that aid inthese functions include transcellular diffusion
andtransport via solute carriers, receptor-mediated trans-cytosis,
and adsorptive endocytosis.
1. Transcellular Diffusion. Early work by Davsondemonstrated
that lipid solubility and size determinedpartitioning of substances
from blood into CSF andbrain tissue (Davson, 1955; Davson and
Smith, 1957). Itis now appreciated that small, lipophilic molecules
cancross the BBB by passive transmembrane diffusion.Passive
transmembrane diffusion is a nonsaturablemechanism by which most
drugs that can enter the
282 Erickson and Banks
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CNSdo so. The degree to which lipid-soluble compoundsmay cross
the BBB is often determined by theirpartitioning into aqueous
versus nonpolar medium,such as water and octanol (Oldendorf, 1974;
Levin,1980). However, as substances that passively diffuseacross
the BBB must traverse the luminal membrane,cytosol, and then the
abluminal membrane prior toreaching the CNS, there is a limit to
the lipophilicity of asubstance that favors diffusion across the
BBB versussequestration within the cell membrane (Banks,
2016).Transcellular diffusion of substances across the BBB isalso
affected when the test substance is an effluxtransporter substrate.
In this case, CNS uptake is muchlower than what would be predicted
based on size andlipophilicity.2. Blood–Brain Barrier Transport via
Solute
Carriers. Solute carrier proteins are integral mem-brane
proteins that permit the directional or facilitateddiffusion of
aqueous molecules across cell membranes.The initial studies that
identified carrier-mediatedtransporters at the BBB investigated
nutritional sub-strates of the brain. D-glucose was the first
substancedemonstrated to have a saturable transport system(Crone,
1965). In this study, Crone used an indicatordiffusion technique
(IDT) to quantify first-pass uptakeof glucose by the brain. The IDT
involves coinjecting aradioactive test substance and a
capillary-impermeanttracer (e.g., Evan’s blue albumin), which
estimates thedilution of the injected substance in blood. The
sub-stances are injected into the carotid artery, and thenvenous
blood from the superior sagittal sinus is imme-diately sampled to
determine the percent loss of injectedsubstance. Crone used the IDT
to assess the brainuptake of D-glucose during hypo- and
hyperglycemicstates, and found that CNS uptake of the glucose
tracerwas highest when blood glucose was low, and lowestwhen blood
glucose was high, indicating that glucoseused a saturable transport
system (Crone, 1965). Sub-sequently, Oldendorf (1971) used a
different techniqueto assess the brain uptake of glucose and amino
acids.The method, called the brain uptake index (BUI), iscarried
out by coinjecting a highly brain-penetrantradioactive standard
along with a radioactive test sub-stance into the carotid artery,
and then immediatelyremoving and counting brain tissue for uptake
of thetest substance with reference to the standard. The BUIis
therefore also a first-pass measurement of brainuptake. In support
of the findings by Crone (1965),Oldendorf’s BUI method also
demonstrated saturabletransport of glucose, as well as amino acids.
Impor-tantly, Oldendorf (1971) also conducted
cross-inhibitionstudies and found that amino acids with similar
chem-ical properties often used a common transporter. It isnow
understood that there are three broad classes ofamino acid
transporters with many subtypes in eachclass (Hawkins et al., 2006;
Abbott et al., 2010). Glucosetransport across the BBB is mediated
by GLUT1 (Dick
et al., 1984; Pardridge et al., 1990; Boado and Par-dridge,
1994), andGLUT1 expression is considered to bea hallmark of BBB
endothelial cells (Cornford et al.,1993, 1994), as its expression
is absent from endothelialcells within brain regions that lack an
endothelial BBB(Rahner-Welsch et al., 1995). In addition to glucose
andamino acids, solute carriers transport nucleosides,
ions,prostaglandins, and many other small polar molecules(Bito et
al., 1976; Abbott et al., 2010). Therefore, onefunction of solute
carriers is to provide the brain withthe essential circulating
substrates for energy genera-tion, protein and nucleic acid
synthesis, and mainte-nance of pH and electrolytes. At the choroid
plexusepithelium, solute carriers are necessary for the pro-duction
of CSF from plasma ultrafiltrate (Johansonet al., 2011). Solute
carrier proteins may be present onthe luminal and/or abluminal
membrane of brainendothelial and epithelial barrier cells, and the
di-rectionality of their transport may be in the blood-to-brain or
brain-to-blood direction, depending on theirorientation within the
membrane (Abbott et al., 2010).
3. Blood–Brain Barrier Transport via Receptor-Mediated
Transcytosis. Another mechanism of trans-port across the BBB is
receptor-mediated transcytosis(RMT), which is thought to be the
predominant mode oftransport of larger macromolecules such as
peptidesand proteins across the BBB (Bickel et al., 2001). RMTis
energy dependent, saturable, and depends on vesic-ular pathways.
RMT may be in the blood-to-braindirection, or brain-to-blood
direction. For ligands tocompletely cross the BBB via RMT, they
must first bindtheir transporter at either the luminal or
abluminalendothelial membrane. Second, they must be internal-ized
in a vesicle, which may be clathrin or cavaeolaedependent
(Georgieva et al., 2014). Third, they must berouted from the
luminal to abluminal membrane orvice versa, which may involve
subcellular traffickingthrough organelles such as endosomes, or the
Golgi(Bickel et al., 2001). Furthermore, ligands must
escapevesicular routing to and degradation by the lysosome.Ligands
may become dissociated from their receptorsduring subcellular
routing, due to the slightly acidic pHof the endosome (Bickel et
al., 2001). Fourth, the ligandmust be exocytosed to the opposite
side of the mem-brane and released into brain interstitial fluid
(Bickelet al., 2001). This final step also requires the
dissocia-tion of the ligand from its transporter. RMT at the BBBcan
be regulated at the level of transporter expression,localization,
and conformation, as well as by concentra-tions of other molecules
that might compete with theligand for transport, or that may
sequester the ligandfrom interacting with its transporter at the
BBB.
4. Blood–Brain Barrier Transport via AdsorptiveTranscytosis.
Adsorptive endocytosis is a receptor-independent mode of vesicular
transport across theBBB and involves interactions of cationic
proteinresidues with the anionic glycocalyx, which lines the
BBB and Neuroimmune Axes 283
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lumial surface of endothelial cells, or membrane glyco-proteins
(Broadwell et al., 1988; Villegas and Broad-well, 1993). Conferring
a positive charge to proteins thattypically do not cross the BBB,
such as albumin (Griffinand Giffels, 1982; Kumagai et al., 1987),
enhances theiruptake by BECs by adsorptive mechanisms.
Adsorptiveendocytosis may be saturable (Kumagai et al., 1987),but
may also be induced by compounds such as wheatgerm agglutinin and
the human immunodeficiencyvirus (HIV) coat proteins TAT and gp120
(Mann andFrankel, 1991; Banks et al., 1998a). Adsorptive
endo-cytosis increases in vitro following lipopolysaccharide(LPS)
or cytokine treatment (Schenk and de Vries,2016).
C. From Brain Barriers to Brain Interfaces:Components of the
Neurovascular Unit
Brain barriers are uniquely poised to communicatesignals between
the CNS and peripheral compart-ments. Communication is not only
achieved throughtransporters, but also frommolecules that are
producedand secreted by cells of the brain interfaces.
Thesesecreted substances can engage autocrine targets,and/or signal
to other cells of the neurovascular unit(NVU), and distal cells in
the brain and periphery. BBBsecretions may be constitutive or
inducible, and are alsopolarized in that they may be released into
either bloodor brain compartments (Banks, 2016). Finally, cells
ofthe BBB respond to signals that arise from the CNS orblood
compartments, which may stimulate alterationsin their barrier,
transport, and secretory functions(Verma et al., 2006; Krasnow et
al., 2017). How theseinterface functions contribute to the
neuroimmunomo-dulatory activities of brain barriers will be
discussed insection II of this review. First, we consider the
individ-ual components and their functions (Fig. 1).1. Endothelial
Cells. In addition to their barrier,
transport, and interface functions, BECs contribute tothe
specialized phenotypes of other cells of the NVU.Endothelial cells
induce astrocyte differentiation invitro via leukemia-inhibitory
factor production (Miet al., 2001). They influence the localization
of the waterchannel aquaporin 4 on the plasma membrane ofastrocyte
endfeet and stimulate the upregulation ofantioxidant enzymes within
astrocytes (Abbott, 2002).Endothelial cells secrete factors such as
transforminggrowth factor (TGF)-b and platelet-derived growthfactor
subunit B (PDGF-B) and signal through Tie2and sphingosine-1
phosphate, which maintain pericytefunctions (Armulik et al., 2005).
Neuroimmune func-tions of BECs are discussed extensively in
latersections.2. Brain Pericytes. Pericytes have important
func-
tions in the development and maintenance of thevascular BBB. Of
the cells of the NVU, pericytes arethe most closely apposed to
capillary endothelial cells;they share a basement membrane and make
direct
contact with BECs via peg and socket as well as gapjunctions
(Dore-Duffy and Cleary, 2011). Brain peri-cytes are derived from
the mesoderm and neuroecto-derm (Winkler et al., 2011) and undergo
proliferativeexpansion and recruitment to the developing
neuro-vasculature during embryonic development and theearly
postnatal period (Daneman et al., 2010). Pericyteattachment to BECs
during embryonic developmentfacilitates BBB tightening by
downregulating genesthat are associated with pinocytic vesicle
formationand immune cell recruitment (Daneman et al., 2010;Ben-Zvi
et al., 2014). PDGF-B produced by braincapillaries signals to
platelet-derived growth factorreceptor b (PDGFRb) on brain
pericytes and regulatespericyte proliferation, attachment to
endothelial cells,and survival. The absence of PDGF-B or PDGFRb
islethal in mice (Leveen et al., 1994; Kaminski et al.,2001),
whereas mice with partial PDGF-B or PDGFRbdeficiency survive into
adulthood, but have reductionsin capillary-associated pericytes
(Armulik et al., 2010;Bell et al., 2010; Daneman et al., 2010).
Pericytedeficiency induced by a PDGF-B mutation results inleakage
of intravascular markers of different sizes intothe CNS, indicative
of BBB disruption. Astrocyteassociations with capillaries were also
altered in thismodel; however, TJ protein expression and
localizationwere relatively unaffected (Armulik et al., 2010).
Micelacking one copy of PDGFRb have an age-dependentloss in
pericytes of about 20% by 1 month of age, and60% by 14–16months
(Bell et al., 2010). BBB disruptionis evident by 1 month and
worsens with age. In thismodel, synaptic deficits and impaired
learning and
Fig. 1. The neurovascular unit. The BBB is in contact and
communicateswith other cells of the CNS as well as circulating
immune cells andperipheral tissues through the endocrine-like
secretions of the latter.Differences occur in NVU function
regionally as well as among theanatomic areas in which barrier
cells are located. As an example of thelatter, immune cell
trafficking occurs largely at the postcapillary venule.Endothelial
cells, astrocyes, pericytes, neurons, and macrophages/micro-glia,
as well as the extracellular matrix and glycocalyx are part of
theNVU. There is renewed interest in mast cell functions, and the
cellulisincompertus represents cell types yet to be discovered that
participate inthe NVU. Not drawn to scale.
284 Erickson and Banks
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memory are evident by 6–8 months, but precede
neuro-inflammation, which does not significantly increaseuntil
14–16 months of age. Pericytes are also importantfor the induction
of the BBB phenotype in vitro, aspericyte coculture with BECs
increases the integrity ofthe barrier (Nakagawa et al.,
2007).Pericytes also have dynamic functions in the NVU.
Pericytes are multipotent stem cells that can differen-tiate
into cells of neural lineage (Dore-Duffy et al.,2006). Theymay also
adopt a contractile phenotype thatcontributes to the regulation of
cerebral blood flow (Hallet al., 2014). Pericytes contribute to the
neuroimmuneresponse and are potent modulators of BBB functiondue to
their proximity to endothelial cells. Pericytessecrete cytokines
and chemokines constitutively inculture and upregulate cytokine and
nitric oxide pro-duction in response to LPS (Fabry et al., 1993;
Kovacet al., 2011). They present antigen in response tointerferon
(IFN)-g, which may contribute to T-cellactivation (Balabanov et
al., 1999). They also enhancethe transcytosis of HIV-1 free virus
and neutrophilsacross in vitro BEC monolayers in the presence of
animmune stimulus (Dohgu and Banks, 2013; Pieperet al., 2013). In
response to injuries that are associatedwith neuroinflammation such
as hypoxia (Gonul et al.,2002) and traumatic brain injury
(Dore-Duffy et al.,2000), pericytes dissociate from the brain
vasculatureand migrate away from the vessels within 1–2
hoursfollowing the insult (Dore-Duffy et al., 2000; Gonulet al.,
2002). In contrast, a systemic inflammatory insultsuch as
intraperitoneal LPS results in pericyte de-tachment from thebasal
lamina between6and24hours,which coincides with reactive
microgliosis and BBBdisruption (Nishioku et al., 2009). Pericytes
that leavethe basement membrane and enter brain parenchymahave been
reported to adopt a phenotype similar to thatof infiltrating
macrophages (Guillemin and Brew,2004). In summary, pericytes may
contribute to theneuroimmune response as follows: 1) causing a
leakyBBB, either by secreting endothelial-disrupting factors,or by
physical disassociation; 2) facilitating the trans-port of immune
cells and pathogens into the brain; and3) propagating
neuroinflammation by stimulating bothresident and recruited immune
cells.3. Astrocytes. Astrocytes are the most abundant
brain cell type and regulate a number of physiologicprocesses in
the CNS that include neurotransmission,synaptic plasticity,
functional hyperemia, and convec-tive flow of brain interstitial
fluid (Sofroniew andVinters, 2010). Astrocytes are also integral in
theinduction and maintenance of the mature BBB pheno-type (Abbott
et al., 2006). Their endfeet surround braincapillaries, arterioles,
and venules. At capillaries, theastrocytic endfeet are located on
the CNS side of thebasement membrane that ensheaths the
endothelialcells and pericytes (Abbott et al., 2006). These
endfeetare in close proximity to the endothelial cells (Thal,
2009) and therefore positioned for crosstalk that pro-motes the
phenotypic specialization of both cell types.The contribution of
astrocytes to BBB formation duringembryonic development is thought
to be negligible inrodents, as astrocytes appear immediately after
birthand do not begin to ensheath brain vessels until the
firstpostnatal week (Daneman et al., 2010). In contrast,radial
glia, which are precursors for neurons andastrocytes, do form
endfeet around capillaries in fetalbaboons and humans (Bass et al.,
1992; Bertossi et al.,1999). Therefore, theremay be species
differences in thecontribution of astrocytes or their precursors to
theembryonic BBB. Astrocytes do contribute to BBBfunctions during
postnatal development and through-out adulthood. In vitro,
astrocytes strengthen thebarrier properties of BECs and also
enhance expressionof BBB transporters, such as Pgp and Glut1,
andenzymes of the metabolic barrier (Abbott et al., 2006).The close
proximity of astrocytes and endothelial cellsat capillaries likely
favors BBB tightening, as in vitrostudies have demonstrated that in
vitro BEC barriersare tightest when astrocytic processes contact
theendothelial monolayer (Abbott, 2002). However, se-creted factors
from astrocytes also contribute to theBBB phenotype. BECs likewise
contribute to the orga-nization of aquaporin 4 and potassium
channels onastrocytic endfeet (Abbott, 2002), which regulate
waterand ion exchange in the CNS (Stokum et al., 2015).Therefore,
communication between endothelial cellsand astrocytes is important
for both the barrier andinterface functions of BECs.
Astrocytes are immune-active cells and were the firstcell type
in the CNS shown to express class II majorhistocompatibility
complex upon IFN-g stimulationin vitro (Wong et al., 1984). More
recent work hasreviewed aspects of astrocyte responses to
systemicinflammation; additionally, CNS injury in the contextof
novel subsets of reactive astrocytes and their func-tions are
beginning to be characterized (Liddelow andBarres, 2017). In
response to systemic inflammatoryinsults such as sepsis, astrocytes
upregulate their pro-duction of proinflammatory cytokines and
chemokines,as well as VEGF (Bellaver et al., 2017).
Inflammatorylesions and interleukin (IL)-1b can induce
astrocyteproduction of VEGF-A and thymidine phosphorylase,which
downregulate TJ protein expression in BECs(Chapouly et al., 2015).
Astrocytes also upregulatecytokines and chemokines in response to
proinflamma-tory stimuli and in disease states (Dong
andBenveniste,2001; Norden et al., 2016). Astrocytic
inflammatoryresponses are differently regulated in comparison
withthose of microglia; for example, astrogliosis
followinginduction of systemic inflammation is inhibited
byindomethacin, whereas microgliosis is not (Bankset al., 2015).
Furthermore, in response to a systemicinflammatory insult,
astrocytes adopt a delayed proin-flammatory phenotype in comparison
with that of
BBB and Neuroimmune Axes 285
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microglia (Norden et al., 2016). Astrocytes may alsofunction in
the resolution of neuroinflammation, as theycan downregulate
microglial activation by secretingTGF-b (Vincent et al., 1997).4.
Neurons. The brain is extensively vascularized—
the mean distance of a neuronal cell body to a capillaryis
approximately 15 mm in mice (Tsai et al., 2009) and30 mM in
nonhuman primates (Mabuchi et al., 2005).Therefore, each neuron
receives and can regulate itsown blood supply from an adjacent
capillary. Neuronsregulate their blood supply through
communicationwith astrocytes, which facilitate dilation of
arteriolesin response to neuronal glutamate release (Zonta et
al.,2003). Some studies suggest that capillary pericytescontribute
to functional hyperemia; however, the rela-tive contribution of
pericytes versus mural cells ofarterioles to neurovascular coupling
has been disputed(Fernandez-Klett et al., 2010; Winkler et al.,
2011;Fernandez-Klett and Priller, 2015). Neuronal activityalso
contributes to the NVU architecture by promotingneurovascular
density and branching during adulthood(Lacoste et al., 2014), but,
during neonatal develop-ment, excessive sensorimotor stimulation
and repeti-tive neural activation result in reduced
microvasculardensity (Whiteus et al., 2014).Neurons exhibit
pleiotropic responses to inflamma-
tory stimuli. Cytokines such as tumor necrosis factor(TNF)-a,
via its interaction with astrocytes, and frac-talkine, which is
expressed by neurons and activates themicroglia fractalkine
receptor, can both stimulate syn-aptic activity (Prieto and Cotman,
2017). Furthermore,cytokines such as IL-1b, IL-6, and IL-18 are
upregu-lated in the brain following long-term potentiation(LTP)
induction in awake rats (del Rey et al., 2013).IL-1b at physiologic
concentrations promotes LTP, butat higher concentrations can
inhibit LTP and impairlearning and memory (Ross et al., 2003;
Prieto et al.,2015). This function of IL-1b may be potentiated
withaging (Prieto et al., 2015). TNF-a is not required forlearning
andmemory, but its overexpression by glia canimpair memory and
synaptic plasticity (Donzis andTronson, 2014).5. Microglia and
Perivascular Macrophages.
Microglia are resident macrophages of the CNS andrapidly respond
to CNS insults. Their lineage is uniquefrom recruited brain
macrophages, in that microgliaderive from the yolk sac during
development, whereasrecruited brain macrophages derive from bone
marrow(Alliot et al., 1999). During development, microgliaassociate
with the brain vasculature and contribute toangiogenesis (Arnold
and Betsholtz, 2013). In adultbrains, microglia remain closely
associated with theneurovasculature. Their production of
proinflammatorymediators such as cytokines, chemokines, nitric
oxide,prostaglandins, matrix proteases, and reactive oxygenspecies
can have profound effects on cells of the NVUand BBB integrity (da
Fonseca et al., 2014). There are
also populations of brainmacrophages that reside in
theperivascular space and are thought to derive from bonemarrow
(Hickey and Kimura, 1988), although morerecent works suggest that
their recruitment to the brainunder physiologic conditions is rare
(Prinz et al., 2011).The perivascular macrophages are thought to
protectthe brain during infection (Polfliet et al., 2001)
andprevent deposition of protein aggregates such as Abpeptide
within the perivascular space (Lai andMcLaurin, 2012). However,
perivascular macrophagesmay also contribute to neurovascular
pathologies asso-ciated with increases in Ab peptide levels in the
brain(Park et al., 2017). An important protective function
ofmicroglia is their ability to rapidly migrate to sites ofbrain
injury and alter their morphology to form aspecialized phagocytic
network that prevents diffusionof harmful substances into the brain
parenchyma (Rothet al., 2014). Microglia also contribute to the
resolutionof inflammation in the brain after injury (Cherry et
al.,2014).
6. Mast Cells. Mast cells are granulocytes thatoriginate from
bone marrow and circulate as precursorcells. Upon recruitment to
tissues, mast cells completetheir differentiation according to
their local environ-ment (Silver and Curley, 2013). Mast cells can
berecruited to the CNS (Silverman et al., 2000; Nautiyalet al.,
2011), where they reside in perivascular spaces ofsome brain
regions, as well as in the choroid plexus andmeninges (Silver and
Curley, 2013). Mast cells areimportant mediators of peripheral
IgE-mediated aller-gic responses; however, they also have emerging
func-tions in the CNS. Granules of mast cells containbioactive
mediators that include histamine, serotonin,serine proteases, and
heparin. Mast cells can alsosynthesize prostaglandins, cytokines,
growth factorssuch as nerve growth factor, reactive oxygen
species,and substance P in response to stimuli (Silver andCurley,
2013). Therefore, mast cells likely play impor-tant roles in
regulating neurotransmission as well asBBB function. Induction of
mast cell degranulationcauses BBB disruption that is localized to
brain regionsenriched in mast cells, such as the medial habenula
indoves (Zhuang et al., 1996). Mast cells are thought tocontribute
to CNS dysfunction in conditions such asstress (Theoharides et al.,
1995), postoperative cogni-tive dysfunction (Zhang et al., 2016),
and rodent modelsof multiple sclerosis (Costanza et al., 2012).
However,mast cells may also regulate physiologic aspects
ofbehavior, as mice that lack functional mast cells in theCNS
display increased anxiety-like behaviors (Nautiyalet al., 2008).
Mast cells are also a predominant source ofbrain histamine
(Goldschmidt et al., 1985). Serotoninderived from mast cells has
been implicated in pro-moting hippocampal neurogenesis and learning
andmemory (Nautiyal et al., 2012).
7. Extracellular Matrix. Within the NVU, basementmembranes
composed of extracellular matrix (ECM)
286 Erickson and Banks
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are present between endothelial cells and pericytes,and also
along the astrocytic endfeet (McConnellet al., 2017). Microvascular
basement membranes arecomposed of laminins, collagen IV,
fibronectin, glycos-aminoglycans including hyaluronan,
chondroitinsulfate-rich proteoglycans, and glycoproteins that
con-tribute to the resiliency of the microvasculature (delZoppo and
Mabuchi, 2003; Lennon and Singleton, 2011;Reed et al., 2017). The
ECM functions as a cellularscaffold that is generated during
development, andendothelial adhesion to the matrix is mediated
byintegrins (del Zoppo and Mabuchi, 2003). Integrins areunique
receptors that can respond to and relay bothintracellular and
extracellular signals (Shen et al.,2012). Blocking the function of
b1 integrin in BECsreduces the expression of the TJ protein claudin
5 andcauses BBB disruption (Osada et al., 2011). The ECM isalso a
barrier to leukocyte and erythrocyte entry intobrain parenchyma
during inflammation and hemor-rhage (del Zoppo and Mabuchi, 2003;
del Zoppo, 2009).Components of the ECM can be degraded by
matrixmetalloproteinases (MMPs), which contribute to BBBdisruption
and leukocyte trafficking during neuroin-flammation (Rosenberg,
2002). MMPs and their modu-latory effects on the BBB have recently
been reviewedelsewhere (Rempe et al., 2016). Hyaluronan and
itsfragments bind to Toll-like receptors, influencing
theneuroimmune environment (Jiang et al., 2011).8. Glycocalyx. The
glycocalyx lines the luminal
surface of endothelial cells, including those of the brain.It is
a gel-like layer estimated to be approximately 5 mmthick, and is
predominantly composed of heparin sulfateproteoglycan, chondroitin
sulfate, hyaluronan, and gly-coproteins (Kolá�rová et al., 2014).
The glycocalyx beginsto form during brain neovascularization during
earlyembryonic development and matures postnatally(Vorbrodt et al.,
1990). The glycocalyx has importantbarrier functions in preventing
direct exposure ofplasma components to the endothelial luminal
mem-brane surface (Vorbrodt, 1989), and it also functions as
amechanosensor and relays signals of sheer stress to theendothelium
(Tarbell, 2010). Degradation of the glyco-calyx occurs during
inflammation, which is associatedwith increased passage of solutes
across the endothelialbarrier, and increased leukocyte adhesion to
the endo-thelium (Kolá�rová et al., 2014; Varatharaj and
Galea,2017).
D. Arms of the Blood–Brain Barrier and TheirNeuroimmune
Functions
The barriers formed by the components above and theresulting
mechanisms by which they form neuroim-mune axes can be categorized
into three main arms: thevascular BBB, the choroid plexus, and the
tanycyticbarrier. The unique cellular and anatomic features ofthese
barriers with reference to their neuroimmunefunctions are discussed
below.
1. The Vascular Blood–Brain Barrier. The vascularBBB is a
broadly applied term that most often refers tothe capillaries
within the brain parenchyma. However,vascular BBBs extend to
pre-and postcapillary arteri-oles and venules, respectively
(Bechmann et al., 2007).Vascular BBBs are also present in the
spinal cord,retina, nerves, and the inner ear, and the structural
andfunctional organization of these has been reviewedelsewhere
(Choi and Kim, 2008). Brain capillariesexhibit functional
heterogeneity within different ana-tomic locations, as certain
brain regions are morevulnerable to disruption during
neuroinflammation(Banks et al., 2015), and transport rates of
immunoac-tive substrates also vary depending on brain
region(Moinuddin et al., 2000; Banks et al., 2001c; Ericksonet al.,
2014). Therefore, the heterogeneity of the vascu-lar BBB imparts
some anatomic specificity to theneuroimmune response.
In brain capillaries, the perivascular space betweenthe
endothelial/pericyte basement membrane andastrocytic endfeet is
small (Thal, 2009). The minimaldistance between capillary
endothelial cells and thebrain parenchyma makes them ideally
positioned forsecreting or transporting molecules into the
CNS(Bechmann et al., 2007). In contrast, precapillaryarterioles and
postcapillary venules have a laminamedia, and the basement
membranes of this layer andastrocytic endfeet form a perivascular
space (Thal,2009). The brain CSF/ISF flows along this
perivascularspace, which facilitates the clearance of solutes from
thebrain parenchyma (Iliff et al., 2012). A subset of
brainmacrophages also resides in perivascular spaces(Bechmann et
al., 2007), and these macrophages arepositioned to respond to
antigens and endogenousimmune signals that are carried by the
perivascularbulk flow of CSF, as well as those that are
transportedor secreted by the endothelium. Transmigration
ofleukocytes across the vascular BBB occurs at postcapil-lary
venules (Owens et al., 2008). Some leukocytes thatcross the BBB
reside in the perivascular space, whereasothers completely
transmigrate across the glia limitansand enter brain parenchyma
(Bechmann et al., 2007).
BECs actively respond to immune stimuli and are anactive
interface in neuroimmune communication.Mech-anisms by which BECs
contribute to neuroimmune axeswill be discussed in section II of
this review.
2. The Blood–Cerebrospinal Fluid Barrier.Epithelial cells of the
choroid plexus are the primarycellular components that
predominantly comprise theblood–CSF barrier/interface. Tanycytes
(see below) andthe arachnoid membrane also interface with the
CSF,but are structurally and anatomically distinct from thechoroid
plexus epithelium (CPE). The choroid plexus isa specialized
structure within all four brain ventricles.Choroid plexuses extend
from the ependymal lining ofthe ventricles, and consist of a single
layer of cuboidalepithelial cells that reside on a basement
membrane.
BBB and Neuroimmune Axes 287
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CPE cells have microvilli and cilia on their apicalsurface,
which contacts the CSF (Damkier et al.,2013). These apical
projections provide a large surfacearea for secretory activity, and
also have functions inregulating and sensing pH, osmolarity, and
ion balancein CSF (Damkier et al., 2013). The basal and
lateralmembranes of CPE cells are relatively flat, except atlateral
membrane contacts near the basal end thatassume a folded labyrinth
structure that is thought tocontribute to a paracellular diffusion
barrier (Damkieret al., 2013). A plexus of leaky blood vessels is
located onthe basal side of the CPE cells. This vascular plexus
isdevoid of astrocytes, and secretions of proteins such asVEGF from
the basal side of CPE cells may contribute tothe fenestrated
vascular phenotype (Esser et al., 1998).Leakage of blood components
into the CSF is preventedby the presence of TJs that are present in
closeproximity to the apical surface of CPE cells (Johansonet al.,
2011). TJ proteins expressed by CPE cells includeclaudins 1, 2, and
11. CPE TJs are thought to be moreleaky than those of the vascular
BBB, and this has beenattributed to the presence of claudin-2,
which can formdiffusive channels within the junction (Amasheh et
al.,2009; Rosenthal et al., 2010; Johanson et al., 2011).However,
the BCSFB is still relatively impermeable assmall molecules such as
ascorbic acid and ions requiresolute carriers for their passage
into CSF (Johansonet al., 2011).The choroid plexus is themajor site
of CSF production
in the brain. Humans produce about 500–600 ml CSFper day, with
approximately 80% of CSF being producedby the choroid plexus and
the remainder derived frombrain ISF (Damkier et al., 2013), as
there is no barrierthat prevents mixing of brain ISF with brain CSF
in theadult (Ghersi-Egea et al., 1996). The CSF provides anutritive
and homeostatic milieu for the brain andcirculates through the
ventricular, subarachnoid, andparavascular spaces in the CNS, as
well as throughbrain tissue, where it mixes with the brain ISF
(Hladkyand Barrand, 2014). This flow pathway contributes tothe
homeostatic milieu of the ISF and also acts as a sinkthat prevents
the localized buildup of solutes in theparenchyma (Oldendorf and
Davson, 1967; Johansonet al., 2011). CSF exits the brain via
arachnoid granu-lations into venous sinuses and in spinal nerves,
as wellas via perineural spaces that penetrate the cribriformplate
(Hladky and Barrand, 2014). CSF turns over inthe human brain
approximately three times per day(Damkier et al., 2013), which is
governed by CSFproduction, convective forces that facilitate bulk
flow,intracranial pressure, and patency of resorption sites(Pollay,
2010). CPE cells express a variety of iontransporters, solute
carriers, and water channels thatcontribute to CSF production by
enabling passage ofwater and solutes from the plasma ultrafiltrate
acrossthe epithelium and into the ventricular space (Damkieret al.,
2013). CPE cells also express efflux transporters
that inhibit the buildup of potentially harmful sub-stances in
CSF. These include ABC transporters such asMRP-1 (Gazzin et al.,
2008) and Pgp (Pascale et al.,2011), as well as monoamine
transporters such asSLC29A4, which facilitates histamine efflux
from CSFinto blood (Usui et al., 2016), and the organic
cationtransporter 3, which clears creatinine from CSF(Tachikawa et
al., 2008). CPE cells also express recep-tors that have been
implicated in transcytosis, includ-ing low-density lipoprotein
receptor-related protein-1(LRP-1), -2, (LRP-2/megalin), and the
receptor foradvanced glycation endproducts (RAGE) (Zlokovicet al.,
1996; Fujiyoshi et al., 2011; Pascale et al., 2011).
The choroid plexus is an immune-active tissue. It is asite of
leukocyte trafficking and immune surveillance(Baruch and Schwartz,
2013; Schwartz and Baruch,2014), and CPE cells upregulate their
expression ofproinflammatory cytokines, c-fos, cell adhesion
mole-cules, and major histocompatibility complex antigensfollowing
systemic inflammatory stimuli (Vallieres andRivest, 1997; Endo et
al., 1998; Wolburg et al., 1999;Engelhardt et al., 2001; Marques
and Sousa, 2015).Structural changes in CPE cells, such as swelling
of theapical microvilli, increases in numbers of dark,
electrondense epithelial cells, and altered mitochondria,
occurduring inflammation (Engelhardt et al., 2001). Inflam-mation
also impairs CSF turnover (Erickson et al.,2012b), which influences
the clearance of solutes fromthe CNS.
The arachnoid epithelium comprises another compo-nent of the
BCSFB. Arachnoid epithelial cells expressTJ proteins such as
claudin 11 (Brochner et al., 2015)and form a size-selective
diffusion barrier in vitro (Lamet al., 2012). Arachnoid epithelial
cells also express highlevels of the efflux transporters Pgp and
BCRP at theirapical membranes (Yasuda et al., 2013), which
areexposed to plasma ultrafiltrate from the leaky vesselsof the
dura matter. Therefore, the activity of thesetransporters would
prevent the transport of substancesfrom blood into CSF in the
subarachnoid space. BCRPexpression was also noted at the basal
membrane ofarachnoid epithelial cells, suggesting that a
secondfunction of BCRP is to facilitate the transport ofsubstances
into CSF (Yasuda et al., 2013).
3. Tanycytic/Ependymal Barriers of Circumventric-ular Organs.
The CVOs of the brain are importantinterfaces for humoral
communication with the CNS.Within CVOs, the brain capillaries are
leaky, lack TJproteins, and are usually fenestrated, allowing
forcommunication of circulating molecules with residentneurons and
glia. The CVOs are generally classified asthose having sensory
functions, which are the subforn-ical organ, organum vasculosum of
the lamina termi-nalis (OVLT), and area postrema, and those
havingsecretory functions, which include the neurohypophy-sis,
median eminence (ME), and pineal gland (Miyata,2015). The
subcomissural organ is also considered a
288 Erickson and Banks
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CVO, but its capillaries are not leaky and express TJproteins
(Petrov et al., 1994; Langlet et al., 2013);however, they lack
GLUT1, which is typically expressedin BBB capillaries
(Rahner-Welsch et al., 1995). Thechoroid plexus (CP) is also
sometimes considered aCVO, but has specialized properties in that
the CPhas an epithelial barrier and is located within the
brainventricles (Miyata, 2015). Neuronal and glial popula-tions
within sensory CVOs can detect and respond tochanges in circulating
components such as electrolytes,glucose, cytokines, and hormones.
In the sensory CVOs,neuronal cell bodies and dendrites are exposed
to blood-derived exudates from leaky capillaries, but projecttheir
axons outside of the CVOs to brain regionsprotected by a BBB
(Rodriguez et al., 2010). In contrast,secretory CVOs receive axonal
projections from neuro-nal cell bodies that are located outside of
the CVO.These axons release peptides into the bloodstream thatcan
signal to distal organs and elicit physiologic re-sponses such as
changes in blood pressure (Mimee et al.,2013). Thus, the CVOs
facilitate bidirectional commu-nication between the brain and
periphery and regulatevital physiologic functions that include
fluid balance,metabolism, reproduction, and immune
responses(Ferguson, 2014).Although serum components can freely
diffuse into
and within CVOs, tanycytic barriers prevent theirdiffusion into
CSF and adjacent regions of the brain.Tanycytes are specialized
ependymal cells that differ-entiate from radial glial cells
beginning in the last fewdays of prenatal development and continue
to maturepostnatally (Edwards et al., 1990). Tanycytes
aremorphologically distinct from cuboidal ependymal cellsthat line
the ventricles in that they lack cilia, andinstead have long,
unipolar projections that are proxi-mal to the fenestrated CVO
capillaries (Rodriguez et al.,2010). Tanycyte structure and
functions have been bestcharacterized in the ME (Mullier et al.,
2010; Rodriguezet al., 2010), although their barrier functions
appear tobe similar in other CVOs (Langlet et al., 2013). Fourtypes
of ME tanycytes have been described, whichinclude a1, a2, b1, and
b2. Barrier properties areascribed to the b1 and b2 tanycytes,
which are locatedat the lateral extensions and floor of the
infundibularrecess, respectively (Rodriguez et al., 2010). The
basalprocesses of b1 tanycytes form bundles with axons thatdefine
the boundary of the arcuate nucleus, which hasan intact BBB, and
the ME (Rodriguez et al., 2010). TJsand AJs are present between
contacts of adjacenttanycyte processes as well as the axons they
surround,and the anatomic location of these b1 projectionsdefines
the diffusion barrier of i.v. injected substancessuch as Evan’s
blue dye between the ME and arcuatenucleus (Rodriguez et al.,
2010). In contrast, the b2tanycytes express TJ proteins, including
ZO-1, occludin,claudin-1, and claudin-5 (Mullier et al., 2010), at
theirapical contacts between cell bodies lining the ventricle
(Rodriguez et al., 2010). These junctions form a barrierthat
prevents diffusion of blood components into theCSF, and similar TJ
organization of tanycytic blood–CSF barriers has been characterized
in the subfornicalorgan, OVLT, and area postrema (Langlet et al.,
2013).Tanycytes of the ME also have important interfacefunctions.
They are thought to participate in the regu-lation of hypothalamic
pathways that control energybalance through glucose sensing and
leptin transportinto CSF (Balland et al., 2014; Elizondo-Vega et
al.,2015).
The sensory CVOs are important interfaces for neuro-immune
communication. Neuroimmune functions ofCVOs were first demonstrated
by the pioneering workof Blatteis et al. (1983) in the 1980s, in
context of thefebrile response, which was diminished by
OVLTablation. It was later shown that cells within CVOsrapidly
upregulate proinflammatory cytokines follow-ing systemic
application of LPS, whereas the brainparenchymal inflammatory
response occurs as a secondwave (Quan et al., 1998). The localized
inflammation inCVOs may disrupt the tanycytic blood–CSF barrier,
asincreased paracellular permeability between junctionsof tanycytes
lining the third ventricle has been observedin response to LPS (Liu
et al., 1996).
III. The Neuroimmune Axes
A. Axis 1: Blood–Brain Barrier Disruption
Strictly speaking, disruption is usually considered apathologic
condition. However, there is a perceptionthat, even under
physiologic conditions, barrier func-tion may slightly vary. As
such, a role for neuroinflam-mation in the physiologic regulation
of barrier tightnessmay emerge, and so this section is presented as
aneuroimmune axis. Furthermore, the term “disruption”as it pertains
to brain barriers is often not clearlydefined. In the strictest
sense, disruption refers to lossof barrier function resulting from
loss of TJ function,reinstitution of macropinocytosis or fenestrae,
ordevelopment of cannulae/vesiculo-tubular structures(Lossinsky and
Shivers, 2004), thus allowing leakageof normally restricted
substances, such as serum pro-teins, across barrier cells. But it
is often used muchmore loosely to describe an alteration in
endothelial orepithelial function permitting leukocyte entry
intobrain parenchyma, loss of Pgp function, or
dysfunctionsresulting in some form of enhanced passage. Notably,the
latter are distinct but possibly interrelated molec-ular processes.
In this work, we will use “disruption” inits strictest sense and
explore inflammatory influenceson immune cell trafficking and Pgp
function in separatesections.
The experimental determination of BBB disruptionand the
subsequent leakage under various neuroin-flammatory states are
often accomplished bymeasuringthe CNS entry of serum proteins or
i.v. injected tracer,
BBB and Neuroimmune Axes 289
-
as well as assessment of TJ protein expression(Saunders et al.,
2015). In living humans, the CSF/se-rum albumin ratio is also a
common indicator used toinfer BBB leakage, and technical aspects of
this ap-proach and others that have been used to detect
BBBdisruption in humans are critically appraised elsewhere(Erickson
and Banks, 2013). BBB leakage is observed indiverse pathologic
states in which distinct modes ofBBB disruption may be apparent. In
this study, wedescribe three mechanisms by which
inflammatoryconditions may contribute to a leaky BBB (Fig. 2).1.
Disruption of Paracellular Tight Junctions.
BBB leakage between endothelial cell contacts canoccur when TJ
proteins decrease in expression, misloc-alize, or are
posttranslationally modified (Luissintet al., 2012). In many
instances, inflammatory media-tors can modulate TJ proteins and
induce paracellularBBB leakage. For example, an injection into
brainparenchyma of IL-1b results in the loss of occludinand ZO-1
expression at endothelial cells, which coin-cides with paracellular
leakage of an intravasculartracer and neutrophil recruitment to
vessels whereTJs are absent (Bolton et al., 1998). TGF-b1,
whichplays classic roles in the resolution of inflammation,also
increases tyrosine phosphorylation of VE-cadherinand claudin-5 and
downregulates claudin-5 expression(Shen et al., 2011; McMillin et
al., 2015). The chemokineC-C motif chemokine ligand (CCL) 2, which
attractsmonocytes, causes loss of TJ protein expression, as wellas
TJ protein redistribution that is mediated by cav-eolae (Stamatovic
et al., 2005, 2006, 2009). Enzymaticdegradation of TJ proteins at
the BBB can also occur inresponse to neuroinflammatory insults. MMP
inhibi-tion or knockout prevents the degradation of TJs andBBB
disruption in the acute phase following brainischemia-reperfusion
injury (Asahi et al., 2001; Yanget al., 2007). Protective factors
have also been identified
that preserve TJ protein expression at the brainendothelium.
These include IL-25, netrin-1, andannexin A1, which are expressed
by BECs, and sonichedgehog, which is secreted by astrocytes (Sonobe
et al.,2009; Alvarez et al., 2011; Cristante et al., 2013;Podjaski
et al., 2015). IL-1b can decrease sonic hedge-hog expression (Wang
et al., 2014b). Other inflamma-tory mediators of increased
paracellular BBBpermeability include bradykinin, histamine,
serotonin,arachidonic acid, and ATP (Abbott, 2000). Clearly, TJsmay
become dysfunctional under a number of differentproinflammatory
states. However, other routes of BBBdysfunction that result in
leakiness also occur in re-sponse to inflammation and are discussed
below.
2. Transcytotic Vesicular Pathways. Ultrastruc-tural studies of
the BBB dating as far back as the1970s have revealed that damaged
BECs can formpatent vesicular channels that permit the passage
oflarge molecular tracers such as horseradish peroxidaseinto the
CNS (Lossinsky and Shivers, 2004). Suchvesicular structures have
been described in cerebraledema, traumatic brain injury, and sepsis
(Castejon,1980, 1998; Esen et al., 2012). In some instances,
theformation of transcellular channels occurs in the ab-sence or
independently of ultrastructural changes inTJs (Lossinsky and
Shivers, 2004; Esen et al., 2012;Goncalves et al., 2017). Despite
the potential contribu-tion of vesicles and transendothelial
channels to BBBleakage, the molecular underpinnings of their
forma-tion remain relatively understudied. One protein that
isubiquitously expressed in peripheral endothelial cells
isplasmalemma vesicle-associated protein (Plvap), whichis sometimes
referred to as MECA-32. Plvap associateswith endothelial fenestrae
and contributes to fenestraeformation in peripheral vessels
(Herrnberger et al.,2012a,b). Plvap is suppressed in brain
endothelium(Hallmann et al., 1995), but is upregulated in
diseases
Fig. 2. Axes 1 and 3: disruption, transport, and penetration.
Major influx mechanisms are transcellular diffusion and saturable
transport. Influx iscountered by efflux (transcellular diffusion,
saturable transport, reabsorption of CSF) and enzymatic activity at
the BBB. Disruption can be by way oftranscellular/transcytotic or
paracellular mechanisms. Endothelial damage and hemorrhage are not
depicted. The extracellular pathways arerelatively inefficient
routes of CNS uptake vs. saturable transport and used by substances
that include albumin, immunoglobulins, erythropoietin, andsoluble
receptors.
290 Erickson and Banks
-
that are associated with BBB disruption, such asAlzheimer
disease (AD) or multiple sclerosis (MS)(Engelhardt et al., 1994; Yu
et al., 2012). Furthermore,Mfsd2a (described above in Features and
Functions ofthe Blood–Brain Barrier and Blood-Brain Interface)
isdownregulated in a mouse model of intracerebralhemorrhage that,
in part, mediates BBB disruption(Yang et al., 2017). Therefore,
formation of endothelialfenestrations may be an important
contributor to BBBleakage in some disease states.3. Endothelial
Cell Damage and Hemorrhage.
Cerebral microbleeds (CMB) are associated with neuro-vascular
insults such as ischemia-reperfusion injury,intracranial
hemorrhage, cerebrovascular diseases, andfollowing traumatic brain
injury (Kleinig, 2013). CMBsare also observed in sepsis patients
(Correa et al., 2012),and systemic inflammation is higher in
patients withCMBs (Miwa et al., 2011). CMBs are visualized
ashemodesmerin deposits, which are iron-rich breakdownproducts of
hemoglobin (Kleinig, 2013). Recent workusing animal models of
subchronic systemic inflamma-tion and ischemia-reperfusion injury
has demonstratedthat BBB disruption and neuroinflammation can
beassociated with subsequent development of microbleeds(Krueger et
al., 2015; Sumbria et al., 2016). In C57BL6/Jmice treated with
three repeated doses of LPS, CMBsbecame evident 2 days after the
final injection andpersisted by day 7. Furthermore, CMBs
significantlycorrelated with markers of neuroinflammation
follow-ing LPS treatment (Sumbria et al., 2016). In a
rodentischemic-reperfusion injury model, it was observed
thatleakage of albumin within the ischemic area occurs inthe
absence of changes in TJ or AJ protein-stainingpatterns (Krueger et
al., 2015). However, structuralalterations to the endothelial
surface were evident andindicated regions where the endothelium was
damagedor absent; these damaged endothelial cells colocalizedwith
albumin extravasation. Ultrastructural analysisrevealed that, at
early stages of damage, endothelial celledema occurs without
apparent extravasation of in-travascular tracer. Influx of tracer
into the brainparenchyma only became apparent after complete lossof
endothelial cell integrity, and influx of red blood cellsinto the
CNS occurred following basement membranedegradation (Krueger et
al., 2015). Neutrophil-derivedproteases such as MMP9 and elastase
have been shownto contribute to the breakdown of the ECM and
de-struction of the endothelium in ischemia-reperfusioninjury
(Gidday et al., 2005; Stowe et al., 2009; Ikegameet al., 2010;
Turner and Sharp, 2016). Intracerebralinjection of neutrophil
elastase causes endothelialswelling and focal necrosis of blood
vessels, as well asfocal hemorrhages and leukocyte cuffing of the
vessels(Armao et al., 1997).The existence of these three modes of
BBB disruption
necessitates a careful interpretation of TJ proteinexpression
data. The absence of apparent changes in
TJ proteins does not necessarily mean that the BBB isintact, as
leakage may occur via vesicles, transcellularchannels, or damaged
endothelial cell membranes.Conversely, a decrease in TJ protein
expression mayreflect BBB damage that is more severe than
para-cellular leakage, such as endothelial cell degenerationwhere
the whole cell is lost in addition to the TJ. Theworks that have
highlighted these varied modes of BBBdisruption also suggest that
different therapeutic ap-proaches to protect the BBB may need to be
consideredbased on which mode of BBB leakage predominates.
B. Axis 2: Modulation of Barrier and InterfaceFunctions by
Immune Substances
The BBB has many functions other than that offorming a barrier
between the peripheral circulationand the CNS. It broadly serves
other roles, includingthat of regulating the homeostatic
environment of theCNS, supplying the nutritional needs of the CNS,
andbeing the center point in the humoral-based communi-cations
between the CNS and peripheral tissues. One ofthe main ways in
which the BBB fulfills these functionsis through the possession of
various transport systems.These transporters have in common that
they are self-saturable. As described above, transporters can
bevariously classified as energy requiring (active trans-port) or
not energy requiring (facilitated diffusion).Active transport can
be unidirectional, transporting asubstance against its
concentration gradient, whereasfacilitated diffusion is
bidirectional with net transportbeing in the direction of higher to
lower concentration.As a rough guide, substances that are the major
ligandfor a blood-to-brain (influx) transporter can enter thebrain
at rates that are 10–100 times faster than if theywere to depend on
nonsaturable mechanisms, andsubstances transported out of the CNS
(efflux trans-porters) accumulate at rates 1/10 or so lower
thanwouldbe expected from their transcellular diffusion.
Severalimportant transporters are modulated by neuroinflam-mation
and neuroimmune substances (Fig. 3). Pgp is amajor efflux
transporter for small, lipid-soluble mole-cules. It resides in the
luminal membrane of BECs andthe ependymal cells forming the choroid
plexus. Asdiscussed above, its ligands represent a diverse group
ofsubstances and include protease inhibitors, opiates(endogenous
and exogenous; peptides and small mole-cules), anti-epileptics,
cyclosporins, glucocorticoids, al-dosterone, dexamethasone, and
calcium channelblockers (Begley, 2004). Its activity explains why
cer-tain substances do not accumulate in the brain insufficient
quantities to produce an effect on the CNS.As such, it can be
viewed either as protecting the brainfrom xenobiotics, including
drugs, that would otherwiseproduce significant CNS side effects or
as a majorobstacle to the development of CNS therapeutics.
Pgpfunction is modulated by inflammation with the maineffect in
vivo being a downregulation of its transport
BBB and Neuroimmune Axes 291
-
function. Details of immune regulation of Pgp functionare
detailed later in section IV of this review.Influx transport can
also be affected by inflammation.
The Na-K-Cl cotransporter at the BEC, important incerebral ionic
homeostasis, is modulated by IL-6 se-creted from astrocytes (Sun et
al., 1997). Insulin istransported across the BBB and acts in the
brain,having effects on cognition and feeding (Banks et al.,2012b).
CSF/serum ratios of insulin are reduced in AD,and delivery of
insulin to the brain of AD patients canimprove cognitive functions
(Craft et al., 1998, 1999,2012). LPS acts indirectly through a
nitric oxide–dependent pathway to increase BBB transport of
in-sulin (Xaio et al., 2001).Some of the many and diverse effects
of LPS on
barrier functions are directly mediated by the presenceof
Toll-like receptors on barrier cells. The expression byBECs of at
least some of these receptors are themselvesregulated by oxidative
stress and TNF-a (Nagyosziet al., 2010). The bacterial cell wall
components LPSand muramyl dipeptide also regulate the BEC
expres-sion of nucleotide-binding oligomerization domain(NOD) and
NOD-like receptors (NLRs), intracellularsensors of pathogen and
damage/danger-associatedmolecules (Nagyoszi et al., 2010).
Expression of NLRsand of NODs, the domain of NLRs that binds
glycopep-tides such as N-acetylglucosamine, is upregulated aswell
by inflammatory cytokines, including IFN-g, TNF-a, and IL-1b
(Nagy}oszi et al., 2015).The response of the brain barriers to
neuroimmune
stimuli can be modulated, reversed, or blocked by anumber of
agents as well. BECs express cannabinoidtype 2 receptors. Agonists
of these receptors prevented32 of 33 genes from being upregulated
by TNF-a anddiminished TNF-induced BBB disruption and
macro-phagemigration (Persidsky et al., 2015). Prostaglandins,
as evidenced by the effects of treatment with indometh-acin, can
block, enhance, or have no effect on the actionsof LPS on BBB
functions (Guillot and Audus, 1990;Minami et al., 1998; Xaio et
al., 2001).
C. Axis 3: Transport, Penetration, and Uptake
ofNeuroimmune-Related Substances
Blood-to-brain entry has been assessed for someneuroimmune
substances. Several cytokines have beenshown to cross the BBB by
way of saturable transportsystems and antibodies and soluble
receptors can enterthe brain by way of the extracellular
pathways.
Many cytokines are transported across the BBB inthe
blood-to-brain direction (Fig. 2). Such transportintermingles the
peripheral pool of the cytokine withits CNS pool. The transport
systems for cytokines aresaturable and are selective, perhaps even
specific, for acytokine or family of cytokines. For example, the
trans-port of TNF-a is self inhibited, but not inhibited by IL-6nor
by any of the IL-1s (Gutierrez et al., 1993; Bankset al., 1994).
The IL-1s (IL-1a, IL-1b, IL-1 receptorantagonist) both self inhibit
as well as inhibit eachother’s transport, but have not been found
to inhibit thetransport of any other cytokine (Banks et al.,
1991).Therefore, the IL-1 family either shares a single
trans-porter or a family of closely related transporters.Epidermal
growth factor crosses the BBB using atransporter shared with TGF-a
(Pan and Kastin,1999). CCL2 (monocyte chemoattractant protein
1)transport is not shared with CCL3 (macrophage-inhibitory protein
1-a) (Ge et al., 2008). Other cytokinesformally demonstrated to be
transported across theBBB include ciliary neurotrophic factor and
TGF-b2(Pan et al., 1999; McLennan et al., 2005), but TGF-b1 isnot
transported across the intact BBB (Kastin et al.,2003). To date,
only IL-2 and CCL11 have been found to
Fig. 3. Axis 2: modulation of barrier/interface function.
Immunoactive (IA) substances work through four main pathways to
alter BBB functions. (A) IAsubstances act on a peripheral cell that
then releases a substance that acts on the barrier. Example: LPS
acts on a peripheral cell inducing it to releasenitric oxide, and
the nitric oxide then acts on BECS to alter insulin transport. (B)
IA acts on the BEC to induce an alteration mediated
throughintracellular machinery. Example: TNF alteration of Pgp
function, which is mediated through a pathway involving nitric
oxide and endothelin-1. (C) IAacts directly at a BEC receptor or
transporter. Example: IL-1ra blocks BBB transport of IL-1b. (D) IA
acts on barrier cell receptor/transporter (i)inducing barrier cell
secretion that acts in autocrine fashion to affect barrier function
(example: LPS induces BEC to secrete IL-6 and
granulocyte-macrophage colony-stimulating factor, which mediates
LPS-induced increase in HIV-1 passage across the BBB) or (ii) to
induce barrier cell tocommunicate with another CNS cell whose
release modifies barrier cell activity (example: presence of
pericyte enhances LPS-induced increase in HIV-1passage across the
BBB).
292 Erickson and Banks
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have a saturable component to their brain-to-bloodefflux phase
(Banks et al., 2004b; Erickson et al.,2014), although others can
enter the circulation withCSF reabsorption (Chen et al., 1997; Chen
and Reichlin,1998). It has also been suggested that the duffy
antigenchemokine receptor on brain endothelial cells contrib-utes
to brain efflux of CCL2 and CCL5 following aneuroinflammatory
stimulus (Minten et al., 2014).Little is known about the cellular
biology of cytokine
transport across the BBB or what circulating factorsmay affect
such transport. Cytokine-induced neutrophilchemoattractant 1 enters
the brain by a nonsaturablemechanism, presumably that of
transcellular diffusion(Pan and Kastin, 2001a). At a mol. wt. of
7800 Da, thiswould be the largest known substance to use
thispathway, which essentially involves the molecule
firstpartitioning into the lipids of the barrier cell membraneand
eventually back into the aqueous environment ofbrain interstitial
fluid or CSF. Erythropoietin entersthe CNS by the nonsaturable
process of the extracellu-lar pathways, although it is unclear the
degree to whichthis underlies its many neuroprotective effects
(Brineset al., 2000; Banks et al., 2004a). In general,
theextracellular pathways account for little of the uptakefor
cytokines that use a saturable transporter to crossthe BBB (Plotkin
et al., 1996). For most endogenousbiologics studied to date, the
protein responsible fortransport is not the same as that used by
the cell forreceptor functions (Pan and Kastin, 1999),
althoughthere seem to be more exceptions to this rule forcytokines
than for other biologics (Pan and Kastin,2002; Pan et al., 2006a;
Ge et al., 2008). CCL2 transportis caveolae dependent (Ge et al.,
2008); IL-2 blood-to-brain transport is inhibited, in addition to
other mech-anisms, by protein binding (Banks et al., 2004b);
andCCL11 (eotaxin-1) binds to cellular components in bloodthat
slows its early-phase entry (Erickson et al., 2014).Most