Three or more routes for leukocyte migration into the central nervous system

© 2003 Nature Publishing Group

In the past, the central nervous system (CNS) has beencharacterized as an immunologically privileged site,but it should more accurately be viewed as animmunologically specialized site. Immune reactions dooccur in the CNS, but take on a distinctive character,which is probably dictated by the local anatomy. In thisregard, the important anatomical features of the CNSinclude: a relative lack of lymphatic drainage of theparenchyma, the lack of endogenous antigen-present-ing cells (APCs), and the BLOOD−BRAIN BARRIER (BBB),which limits the exchange of immune cells and media-tors. Because the CNS can deploy only a limited arrayof immune-defence components, researchers havebeen challenged to define the specific mechanisms thatsupport immune reactivity in the CNS. This topic haswide-ranging and considerable clinical implications.Some immune reactions are suspected of being delete-rious, as in the case of the inflammatory demyelinatingdisease multiple sclerosis (MS), and possibly thecytokine environment that is produced in response tothe chronic presence of HIV-1 in MICROGLIA1. Conversely,in response to tumours of ASTROGLIA, the immune reac-tion in the brain seems inadequate to protect the host2. Inother circumstances, such as the inflammatory responseto plaques in Alzheimer disease, the inflammatory

reaction might be insufficient to clear noxious mater-ial, but might still result in the production of toxic by-products3. The importance of understanding themechanisms of CNS immune reactivity is highlightedby the recent termination of a promising clinical trialof the Alzheimer disease vaccine AN-1792 after thedevelopment of encephalitis in at least 15 study partici-pants4. Elucidating the molecular pathways by whichleukocytes enter the CNS is crucial to understandingintrathecal immune reactivity. Advances in decipheringthe trafficking of leukocytes to small intestine, skinand lymph nodes provide a model for addressing howhaematopoietic cells migrate throughout the body inhealth and disease. Building on this progress, there isa large and increasing amount of experimental litera-ture that addresses trafficking of haematopoietic cellsto the CNS. However, to obtain a coherent view ofCNS inflammation, it will be necessary to place thisdata in the context of the local vascular and compart-mental anatomy. Here, we review the anatomical fea-tures of the CNS, interpret existing data in the lightof these considerations and discuss the most com-pelling issues that are outstanding in the search for acomprehensive account of the inflammatory responsein the CNS.

THREE OR MORE ROUTES FORLEUKOCYTE MIGRATION INTO THE CENTRAL NERVOUS SYSTEMRichard M. Ransohoff*, Pia Kivisäkk and Grahame Kidd

Leukocyte migration into and through tissues is fundamental to normal physiology,immunopathology and host defence. Leukocyte entry into the central nervous system (CNS) is restricted, in part, because of the blood−brain barrier (BBB). During the past decade, crucialcomponents that are involved in the process of leukocyte migration have been identified andprogress has been made in understanding the mechanisms of neuroinflammatory reactions. In this review, present knowledge of the trafficking determinants that guide the migration ofleukocytes is superimposed onto the vascular and compartmental anatomy of the CNS. Wediscuss three distinct routes for leukocytes to enter the CNS and consider how differentpopulations of leukocytes use trafficking signals to gain entry.

NATURE REVIEWS | IMMUNOLOGY VOLUME 3 | JULY 2003 | 569

Department ofNeurosciences, The LernerResearch Institute and *The Mellen Center forMultiple Sclerosis Treatmentand Research, Departmentof Neurology, 9500 EuclidAvenue, The ClevelandClinic Foundation,Cleveland, Ohio 44195, USA.Correspondence to R.M.R.e-mail: [email protected]:10.1038/nri1130

BLOOD-BRAIN BARRIER

(BBB). The physiologicalbarrier that separates bloodfrom brain parenchyma.It consists of endothelial cells with tight junctions that aresurrounded by a continuousbasement membrane andastroglial end-feet.

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570 | JULY 2003 | VOLUME 3 www.nature.com/reviews/immunol

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and implications of leukocyte recruitment across thedifferent vascular beds and to various fluid compart-ments of the CNS will differ, and need to be distin-guished to allow the precise articulation and evaluationof hypotheses. The phrase ‘leukocyte entry into theCNS’ is commonly used in published research reportsthat deal with this topic. But given the complexity ofpossible mechanisms, this term is so imprecise as to bevirtually meaningless.

The unique CNS anatomyThe CNS is comprised of the brain and spinal cord,encased in bone (either skull or vertebral column) anda threefold membranous covering — the outermostdural membrane, the intermediate arachnoid membraneand the innermost pial membrane (FIG. 1). As it is pro-tected, nourished and supported by a unique and diversecombination of vascular elements and by the cere-brospinal fluid (CSF), the mechanisms, consequences

Meningeal vesselsVenous sinus

Skull bone

Dural membrane

Arachnoid membrane

Pial membrane

Ventricles

Choroid plexus

Grey matterWhite matter

Subarachnoid space

CSFflow

Dural membrane

Arachnoid villi

Arachnoidmembrane

Subarachnoid space

Pial membrane

Glial limitans

Cerebral cortex Falx cerebri

Venous sinus

CSF flow

Perivascular space(Virchow–Robin space)

Figure 1 | Anatomical structures involved in the arterial supply of the CNS and the cerebrospinal fluid circulation. Thearterial supply of the brain parenchyma is derived from terminal branches of the internal carotid arteries following the brain surface inthe subarachnoid space. When entering the brain parenchyma, the vessels are initially surrounded by the perivascular space (alsoknown as the Virchow−Robin space), which is connected to the subarachnoid space (insert). Arterial supply is also provided bydeep penetrating branches from the internal carotid artery. The cerebrospinal fluid (CSF) is actively secreted by the choroid-plexusepithelium that is located in the ventricular system of the brain. CSF circulates from the ventricles to the subarachnoid space locatedbetween the arachnoid and the pial membranes, and is resorbed to the systemic circulation through the arachnoid villi that extendinto the venous sinuses of the cerebral hemispheres.

MICROGLIA

Interstitial cells of mesodermalorigin that form part of thesupporting structure of thecentral nervous system. Theyhave a migratory capacity andfunction as phagocytes of thenervous tissue.

ASTROGLIA

Star-shaped cells of ectodermalorigin that provide nutrients,support and insulation forneurons.



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the nasal submucosa9. The physiological relevance ofthis pathway was supported by tracer studies thatshowed that the quantitative contribution of cervicallymphatics to bulk CSF drainage is substantial (esti-mated to be 50%)10. So far, these experiments havefocused, in particular, on the lymphatics that are associ-ated with the olfactory bulbs. However, regional lym-phatics that are associated with other cranial nervesmight also be involved in the drainage of CSF.

It is not entirely certain whether these findings canbe extrapolated to humans. Anatomical studies showedsimilar structural relationships between the arachnoidvilli, the cribriform plate of the ethmoid bone and lym-phatics of the nasal submucosa in humans, as were previously reported in other species. Furthermore, inEXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE) — amodel of inflammatory demyelinating disorders —myelin antigens were detected in the cervical lymphnodes of non-human primates, and these myelinantigens were associated with cells of dendritic-cellmorphology and cell-surface phenotype11.

The ependymal lining of the ventricles lacks TIGHT

JUNCTIONS, placing relatively little impediment betweenthe extracellular fluid of the CNS white matter and ven-tricular CSF. The CNS grey matter interstitial fluid alsoequilibrates with the CSF at the surface of the brainwhere specialized perivascular spaces (also known asVIRCHOW−ROBIN SPACES) that are associated with penetrat-ing arteries are continuous with the subarachnoid space(FIG. 1). Such pathways of clearance of interstitial fluid

CSF flow connects the CNS to lymphatics. The CSF, pre-viously regarded as an ultrafiltrate of plasma, is, in fact,actively produced by the secretory epithelium of thechoroid plexus (CPE)5. Choroid plexuses are located inindividual cavities (known as ventricles) in separateregions of the brain (FIG. 1). CSF circulates from the ven-tricles through the SUBARACHNOID SPACE, which is locatedbetween the arachnoid and the pial membranes, and ismainly resorbed into venous blood through the arach-noid villi, which are ‘outpouchings’ of the arachnoidmembrane that extend into the venous sinuses of thecerebral hemispheres. Formation and absorption of CSFare extensive processes — the human CSF volume turnsover approximately four times each day.

Of interest for immune surveillance of the CNS,both recent and previous studies of rodents and rumi-nants indicate that CSF also drains into cervical lymphnodes6,7. The notion that CSF flows into the cervicallymphatics has been considered somewhat heretical,because it is commonly stated that the CNS lacks lym-phatic drainage. Indeed, lymphatic vessels are not foundin CNS tissue; accordingly, the CSF might be a partialfunctional equivalent of the lymph for the CNS8.Evidence indicating that CSF drains into the lymphaticscame from finding anatomical connections between thesubarachnoid spaces around olfactory bulbs — pairedstructures found above the nasal cavity on the ventralsurface of the brain that receive input from olfactoryreceptors — that drain across the paper-thin cribriformplate in the base of the ethmoid bone into lymphatics of

SUBARACHNOID SPACE

A space between the arachnoidand pial membranes thatsurround the brain and spinalcord that is filled withcerebrospinal fluid. It containsfibrous trabeculae, blood vesselsand antigen-presenting cells.

EXPERIMENTAL AUTOIMMUNE

ENCEPHALOMYELITIS

(EAE). An animal model thatmimics some of the clinical andhistopathological characteristicsof multiple sclerosis. EAE can beinduced in various species byimmunization with myelinantigens or adoptive transfer ofneuroantigen-specific T cells.

TIGHT JUNCTIONS

Intercellular junctions whereadjacent plasma membranes are joined tightly together,occluding the intercellular spaceand limiting the intercellularpassage of molecules.

VIRCHOW–ROBIN SPACE

A space that surrounds bloodvessels for a short distance asthey enter the brain from thecortical surface, defined byextensions of the arachnoid andpial membranes.

Choroid plexus

Brain parenchyma

Subarachnoid space

Peripheral lymph node

Ependyma

a

c

b

d

Systemiccirculation

Virchow–Robin space

Solubleproteins

Bloodvessel

T cell

Myeloid APC

Cerebralventricle CSF

Figure 2 | Afferent and efferent mechanisms of immune surveillance in the CNS. The choroid plexus, cerebral ventricle,subarachnoid space, brain parenchyma, systemic circulation and a peripheral lymph node are shown in cartoon form. Afferentsignals from the CNS parenchyma to the peripheral immune system are initiated by movement of soluble proteins into thecerebrospinal fluid (CSF), either from white matter across the ependyma or from grey matter along the perivascular channels. Fromthe CSF, soluble proteins are transported through lymphatic channels to peripheral lymph nodes and can provide antigenicstimulation to naive or memory T cells. The efferent phases of immune reactions are initiated in secondary lymphoid organs (notshown) and promoted locally by restimulation through interactions between memory T cells and antigen-presenting cells (APCs).Memory T cells are proposed to migrate from blood through the subarachnoid space and back to the systemic circulation asindicated by dotted arrows. APCs of the CNS include a variety of myeloid-lineage cells, which give rise to many sites of potentialefferent immune interaction. Resident APCs of the CNS include: choroid-plexus macrophages (a), epiplexus cells (b), meningealmacrophages (c) and perivascular cells of the Virchow–Robin spaces (d).



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Distinct character of inflammation of the CNSSpecific anatomical features of the CNS seem to haveselected the character of immune and inflammatoryresponses at this site8,16. First, the CNS is encased in rigidbone and is covered by an inelastic dural lining.Therefore, the total volume of the CNS is invariant. Anyincrease in the volume of extracellular fluid, as occurswith inflammatory swelling, will increase tissue pres-sure, oppose arterial influx and threaten secondaryischaemic damage. Second, the function of the CNSdepends on the viability of neurons, which are mainlypost-mitotic and non-regenerating. Given these con-straints, it is perhaps predictable that the CNS has a lim-ited capacity for inflammatory and immune reactivity.In addition to the absence of tissue lymphatics, carefulultrastructural and dual-colour immunostainings haveshown that there is a low level of expression of MHCclass II molecules in the intact human brain17,18.Although it has been controversial previously, it hasrecently been shown that virtually all expression ofMHC class II molecules in the brain parenchyma isrestricted to reactive microglia and phagocyticmacrophages18, cells with limited capacity for antigenpresentation to naive cells, and no resident dendritic-cellpopulation has been detected in the brain parenchyma.

It is not surprising, therefore, that immune andinflammatory responses in the CNS are different fromthose in other internal organs. For example, necrosis inmost solid organs, such as the liver, elicits a strongresponse that is characterized by the recruitment andactivation of monocytes and neutrophils. However,neurotoxin injections in the CNS, which induce markedneuronal necrosis, fail to elicit a typical leukocyteresponse19−21. After neurotoxin challenge, a rapid reac-tion of the resident microglia is observed, but the accu-mulation of monocytes is delayed and, unexpectedly,there is no neutrophil response. Furthermore, previousstudies showed that there was a delayed rejection ofxenogenic tumour implants in the CNS parenchyma22,compared with the skin. More recent experimentsshowed slow and inefficient clearance after inoculationof virus into the parenchyma23. Therefore, the claim thatthe CNS is a site of immune privilege has been modi-fied, and it is now proposed that the CNS is a site ofselective and modified immune reactivity.

Trafficking into CNS vascular compartmentsDespite the successful studies of T-cell homing to gut,skin and lymphoid organs (BOX 1 and TABLE 1), thedeterminants involved in the selective trafficking toother organs are not known. Studies of inflammationin internal organs such as heart, pancreas, joint, lungand CNS have, so far, failed to yield organ-selectivetrafficking determinants. It seems obvious that suchdeterminants should exist, because the challenges ofimmune defence of, for example, the lungs, joints,heart and brain, clearly differ.

It is possible to define three distinct routes of leuko-cyte entry into the nervous system and to evaluatewhat we know about trafficking through these accesssites (TABLE 2). To set an appropriate background for

from grey and white matter of the CNS seem to dealefficiently with soluble proteins12. So, it seems probablethat protein antigens in the CNS can readily accesslymphoid tissues through the CSF and cervical lym-phatics and, after appropriate processing and presenta-tion, can stimulate antigen-specific responses by naiveor memory T cells that express cognate receptors.Furthermore, these observations provide a frameworkfor speculation about the physiological immune sur-veillance of the CNS (FIG. 2). The afferent limb of theCNS immune response is provided by transit of anti-genic material from the parenchyma to the CSF andthen to the cervical lymphatics. The efferent limbmight largely occur in the subarachnoid space. In sup-port of this, leukocytes found in the CSF of healthyindividuals are mainly CD4+ memory T cells13. Thesememory cells circulate through the subarachnoidspace, where they might encounter myeloid cells thatare capable of antigen presentation at several sites: inthe choroid-plexus stroma, associated with theependyma, in the meninges or in the Virchow−Robinperivascular spaces (FIG. 2).

In summary, it is probable that control of immunereactivity to components of the CNS cannot occursolely by sequestering neuroantigens behind the BBB.Rather, trafficking of immunocompetent cells into theCNS must also be tightly regulated.

Blood−brain barrier and blood−CSF barrier. Capillariesof the cerebral vasculature protect the CNS and excludecirculating cells and macromolecules owing to inter-endothelial tight junctions, similar to those that arefound between epithelial cells. These tight junctions pre-vent the movement of leukocytes in or across theendothelium. Trans-cellular movement of solutes is alsoprecluded, as the endothelial cells have a poor capacityfor pinocytosis and endothelial fenestrae are absent.These unique features of the capillary endothelium thatexclude serum proteins and tracer molecules from theCNS are collectively known as the BBB14. The choroidplexus is comprised of the CPE cells that are arrayed invilli, surrounding a core of vascularized stroma. Thecapillaries of the choroid-plexus stroma are fenestrated— having ‘window’-like openings of ~80 nm across thecapillary wall — and lack tight junctions; nevertheless,serum proteins do not freely pass from blood to CSF,due to tight junctions between the CPE cells. Thesetight junctions constitute the anatomical basis of theblood−CSF barrier.

Taken together, the BBB, the blood−CSF barrier andthe CSF circulation exert bi-directional control —blood-to-brain and brain-to-blood — of the passage ofa large diversity of regulatory and nutrient proteins,electrolytes and free fatty acids, as well as toxins. TheBBB is supported in its defence of the CNS fromintravascular toxins by other mechanisms, such as apotent efflux apparatus that promotes the movement ofnoxious substances in the CNS-to-blood direction5.Matching these efflux transporters are blood-to-brain influx mechanisms that supply the brain withnutrients15.



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way seems likely to be one route by which cells enter theCSF in normal physiological conditions. Support forthis concept came from studies in which fluorescence-labelled lymphocytes were injected intravenously intohealthy mice, and 2 hours later were found in thechoroid-plexus stroma (a plausible point on the route)and the meninges24. One insight into the mechanism ofthis migration pathway was indicated by a markedreduction in leukocyte entry into the CNS in mice thatlacked P-selectin.

Relatively little is known about how leukocytesaccess the CSF in other species. However, the leukocytesin the CSF of humans have a distinctive phenotype,which indicates the occurrence of a regulated process.Neutrophils — the main population of circulating

this discussion, it should be known that there are fewleukocytes present in the CNS unless inflammationoccurs. Therefore, most of the research on traffickinginvolves the use of disease or injury models.

Route 1: from blood to CSF across the choroid plexus.The first pathway of migration of leukocytes into theCNS follows the formation of CSF. This route is likely tobe of physiological relevance, as CSF of healthy individ-uals contains ~3,000 leukocytes per ml. In this pathwayof immigration, leukocytes extravasate across the fenes-trated endothelium of the choroid-plexus stroma,migrate through the stromal core to the villi, interactwith epithelial cells of the choroid plexus and enter theCSF at its site of formation (FIG. 3). At present, this path-

Table 1 | Determinants involved in leukocyte trafficking

Selectins Chemokines Integrins ChemokinesCarbohydrate ligands G-protein coupled Adhesion molecules G-protein coupled

receptors of Ig superfamily receptors

Tethering/rolling Activation Adhesion Diapedesis

Lymph nodes L-selectin/PNAD CCR7/CCL21 LFA1/ICAM1 CCR7/CCL19,21

Effector T-cells CLA/E-selectin CCR4/CCL17 LFA1/ICAM1 CCR10/CCL27to inflamed skin

Small intestine α4β7/MADCAM1 ??/?? LFA1/ICAM1 CCR9/CCL25lamina propria

Adhesion molecules, chemokines and chemokine receptors determine patterns of tissue-specific lymphocyte homing. The cartoonshows a leukocyte undergoing the sequential stages of extravasation, through a programmed series of interactions with the endothelium,followed by transmigration across the basal lamina. The molecules implicated in each step of this process are listed. α4β7, integrin α4β7;CCL, CC-chemokine ligand; CCR, CC-chemokine receptor; CLA, cutaneous lymphocyte-associated antigen; ICAM1, intercellularadhesion molecule 1; LFA1, leukocyte function-associated antigen 1; MADCAM1, mucosal vascular addressin cell adhesion molecule 1;PNAD, peripheral node addressin.

Endothelium

Basal lamina

Box 1 | Trafficking determinants for leukocytes

Leukocyte extravasation has been separated into discrete steps, which are associated with interacting pairs of selectinsand their ligands, integrins and cell-adhesion molecules (CAMs), and chemokines and chemokine receptors (TABLE 1).The initial contact between a leukocyte and an endothelial cell is referred to as ‘tethering’, and subsequent interactionsare referred to as ‘rolling’. Both steps are associated with selectins and their carbohydrate ligands. These molecules aredistributed both on leukocyte MICROVILLI, where they can mediate both tethering and rolling, and on the cell soma, whereonly rolling occurs112,113. Furthermore, α4 integrins, but not β2 integrins, can also support rolling114.

The later stages of extravasation require the engagement of a G-protein-coupled receptor expressed by the leukocytewith an appropriate ligand115. For simplicity, this review focuses on the chemokines and their receptors. Chemokinesand chemokine receptors mediate the activation of integrins, which can include both clustering of integrins in the planarmembrane (leading to increased avidity) and modulation of integrin conformation to achieve a state of high affinity forbinding of CAMs116. Interactions between activated leukointegrins and endothelial CAM counter-receptors lead to firmadhesion, arrest and flattening of leukocytes115. The final stage of extravasation is diapedesis, which requires cytoskeletalreorganization, that depends on a second set of signals through chemokines and their receptors117. For a given leukocyteextravasation event, it is possible to identify the following: a rolling determinant (selectin−selectin-binding carbohydratemoiety), a leukointegrin-activator (chemokine−chemokine receptor), a mediator of arrest (leukointegrin–CAM pair)and the chemokine–chemokine receptor interaction that drives diapedesis118,119. TABLE 1 shows the molecular pairs thathave been implicated at different stages of lymphocyte migration into the lymph nodes, inflamed skin and the laminapropria of the small intestine. These examples indicate the level of precision that can be attained in understanding thespecificity of leukocyte trafficking.

MICROVILLI

Small processes or protrusionsfrom the cell surface that increasethe surface size of the cell.



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T HELPER 1 (TH1) CELLS29, is CXCR3 selectively expressed by a

subset of cells that use other mechanisms to enter thiscompartment, or is the expression of CXCR3 upregulatedduring extravasation? As noted earlier, it is probable thatthis population of CD4+ central memory T cells carryout immune surveillance of the CNS during normalphysiological conditions.

It is also clear that a rapid T-cell response to challengecan occur in the subarachnoid space. In an experimentalmeningitis model, adult mice accumulate 1 × 106 leuko-cytes per ml of CSF within 6 hours of intracerebral chal-lenge with Streptococcus pneumoniae30. A stimulus thatdoes not activate TOLL-LIKE RECEPTORS (TLRs) of the innateimmune system is also effective: intraventricular inocula-tion of protein antigen led to the rapid accumulation ofantigen-specific T cells in the CSF of mice that expresseda transgenic T-cell receptor (TCR) specific for that anti-gen. T-cell accumulation was insensitive to antibodiesspecific for vascular-cell adhesion molecule 1 (VCAM1),

leukocytes — are rarely detected in CSF of healthyhumans. Instead, CSF leukocytes are mainly T cells,which constitute ~80% of CSF cells, as compared withno more than 45% in blood25. Furthermore, the ratio ofCD4+ T cells to CD8+ T cells is increased, compared withperipheral blood. Monocytes constitute ~5% of CSFcells; less than 1% B cells. CD4+ T cells in CSF mainlyexpress cell-surface phenotype markers that are expressedby CENTRAL MEMORY T CELLS (CD27 and CD45RO) (REF. 26).Furthermore, the level of expression of very late antigen 4(VLA4) is increased in T-cell populations of the CSF,compared with those in the blood13. Compared withCD4+CD45RO+ T cells in the circulation, CSF T cellsexpress higher levels of CXC-chemokine receptor 3(CXCR3), but similar levels of CCR1, CCR2, CCR3,CCR5 and CCR6 (REF. 27). This finding is descriptive buttantalizing. Does CXCR3 mediate T-cell entry into theCSF? Alternatively, as T-cell activation results in the stableupregulation of expression of CXCR3 (REF. 28), mainly by

CENTRAL MEMORY T CELLS

Previously activated memory T cells that have encounteredantigen in secondary lymphoidorgans and obtained the capacityto migrate through extra-lymphoid tissues, but retainreceptors and ligands, such asCC-chemokine receptor 7 andL-selectin, allowing the cells toreturn to the lymphoidcompartment.

Table 2 | Effects of blockade or genetic depletion of determinants with a possible role in trafficking of leukocytes to the CNS

Encephalito- Interaction with inflamed vessels Lymphocyte Severity of EAEgenicity of migrationT-cell clones into the CNS

Choriod Pial surface Parenchyma Spinal cordplexus

P-selectin

Blockade N.D. N.D. ↓ rolling and ↓ rolling and N.D. ↓ to non- No effect55

adhesion in vivo36 adhesion in vivo38 inflamed brain67

Knockout mice N.D. N.D. ↓ rolling and N.D. N.D. ↓ to CSF during N.D.adhesion in vivo56 meningitis104

E-selectin

Blockade N.D. N.D. No effect in vivo36 ↓ rolling in vivo38 N.D. N.D. No effect55

Knockout mice N.D. N.D. ↓ rolling and N.D. N.D. N.D. N.D.adhesion in vivo56

ICAM1

Blockade ↓101 ↓ adhesion N.D. ↓ arrest in vivo38 N.D. N.D. ↓ aEAE, no effectin vitro103 on tEAE51,105−107

Knockout mice N.D. N.D. N.D. N.D. N.D. N.D. ↑108

LFA1

Blockade N.D. ↓ adhesion N.D. ↓ arrest in vivo38 N.D. N.D. ↑50−52 ↓53

in vitro103

VCAM1

Blockade ↓101 ↓ adhesion N.D. (↓ ) arrest ↓ capture and N.D. ↓101,109

in vitro103 in vivo38 adhesion in vivo39

VLA4/integrin-αα4

Blockade ↓101,102 ↓ adhesion ↓ rolling and arrest ↓ adhesion in vitro42 ↓ capture and No effect, ↓42,43,54,101,109

in vitro103 in vivo during EAE, (↓ ) arrest in vivo38 adhesion in vivo39 non-inflamed but not in pre- brain67

symptomatic mice36

MADCAM1

Blockade N.D. N.D. N.D. N.D. N.D. N.D. ↓110

Integrin- ββ7

Blockade N.D. N.D. N.D. N.D. N.D. N.D. No effect onRR EAE109

↓ chronic EAE111

Knockout mice N.D. N.D. N.D. N.D. N.D. N.D. ↓111

aEAE, active-immunization EAE; CNS, central nervous system; CSF, cerebrospinal fluid; EAE, experimental autoimmune encephalomyelitis; ICAM1, intercellular cell-adhesionmolecule 1; LFA1, leukocyte function-associated protein 1; MADCAM1, mucosal addressin cell-adhesion molecule 1; N.D., not determined; RR, relapsing remitting; tEAE,adoptive-transfer EAE; VCAM1, vascular cell-adhesion molecule 1; VLA4, very late antigen 4.



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exchange with circulating leukocyte populations33−35.Therefore, the perivascular regions, in particular theVirchow−Robin spaces, which are in direct communi-cation with the CSF compartment, are consideredprobable sites of lymphocyte–APC interaction and,therefore, of immune surveillance of the CNS (FIG. 2).Recent studies using intravital microscopy analysedleukocyte rolling on capillaries at the pial surface of thecerebral hemispheres36. Leukocyte rolling on these ves-sels was essentially absent in healthy mice. Rolling andadhesion were both stimulated by inducing EAEthrough immunization with a myelin-protein-derivedpeptide in complete Freund’s adjuvant (CFA) andBordetella pertussis toxin. Leukocyte rolling was par-tially sensitive to antibody-mediated blockade of α4integrin, but absolutely required P-selectin. Adhesionwas only partially blocked by eliminating rolling withantibody specific for P-selectin.

Route 3: from blood to parenchymal perivascular space.In the third pathway, leukocytes can enter the paren-chyma directly, passing from internal carotids, throughthe branching vascular tree of arterioles and capillaries,finally extravasating through postcapillary venules. Inthis case, leukocytes are required to cross the BBB andthe endothelial basal lamina. Previous studies carriedout by Hickey and co-workers34, and separately byWekerle and colleagues37, showed that activated T-cellblasts, after intravenous injection, can undergo thisextravasation event. The efficiency of extravasation wasquite low and, importantly, extravasation by this path-way occurred independently of antigen specificity —mitogen-activated and encephalitogenic neuroantigen-specific T-cell blasts were equally efficient. Relativelylittle is known about the mechanisms by which theseT-cell blasts extravasate. A new strategy of intravitalmicroscopy was recently used to analyse leukocyteextravasation across parenchymal venules during EAE38.In these experiments, intravital microscopy was carriedout through the intact skull, and fluorescence-labelledcells were injected into a common carotid artery afterligation of the ipsilateral external carotid, forcing theinjected cells into the internal carotid and then to cere-bral microvessels. Two cell populations were used: unac-tivated peripheral lymph-node lymphocytes or activatedneuroantigen-specific cells. No leukocyte−endothelialinteraction was observed in healthy mice. Notably,highly activated neuroantigen-specific encephalitogeniclymphocytes failed to interact with the cerebral micro-vessels, unless the vessels were previously activated byintravenous injection of lipopolysaccharide (LPS) oradministration of tumour-necrosis factor (TNF). Thisobservation supported the notion that transit of acti-vated lymphocytes across a resting cerebrovascularendothelium is a low-efficiency event, below the limit ofresolution in these studies.Where leukocyte−endothelialinteractions were observed, antibodies specific for P-selectin or P-selectin glycoprotein ligand 1 (PSGL1)blocked rolling, as described earlier for vessels at the pialsurface36. In the paper by Piccio et al.38, it was furthershown that interactions between integrins and adhesion

intercellular adhesion molecule 1 (ICAM1) or their inte-grin co-receptors. Injection with a platelet endothelial-celladhesion molecule (PECAM)−immunoglobulin fusionconstruct eliminated leukocyte entry into the CSF, consis-tent with a role for PECAM in this model31. Interestingly,PECAM1-deficient mice develop an abnormal BBB, withmarkedly inefficient repair of the barrier after it wasopened by vasoactive amine challenge, both in vitro andin vivo32. Because endothelial PECAM1 was shown by theknockout studies to be necessary for a fully functionalBBB, it seems possible that inhibition of leukocyte migra-tion into the CSF was mediated by homophilic interac-tions between PECAM and PECAM−immunoglobulinfusion protein on the leukocyte plasma membrane.

Route 2: from blood to subarachnoid space. In the secondpathway, leukocytes can traverse from the internal carotidartery,extravasating across postcapillary venules at the pialsurface of the brain into the subarachnoid space and theVirchow−Robin perivascular spaces (FIG. 1). There, theymight encounter cells of the monocyte/myeloid lineagethat are competent for antigen presentation. Using bone-marrow chimaeras, it was shown that perivascular cells

T HELPER 1/2 CELLS

(TH1/T

H2). Activated CD4+

T cells differentiate into twodistinct phenotypes that areassociated with highly polarizedimmune responses. Generally,T

H1 cells produce high levels of

interferon-γ, lymphotoxin andtumour-necrosis factor, and areassociated with cell-mediatedimmunity, whereas T

H2 cells

produce high levels ofinterleukin-4 (IL-4), IL-5 andIL-13, and are associated withhumoral immunity.

TOLL-LIKE RECEPTORS

(TLRs). A family of receptorsthat recognize conservedproducts that are unique tomicroorganisms, such aslipopolysaccharide. Stimulationthrough TLRs inducesmaturation and activation ofdendritic cells, leading tooptimal activation of theadaptive immune response.

Endothelium

Epithelium

StromaChoroidplexus

Cerebrospinal fluid

Venulea b c

d

e

f

Selectin ligandsChemokinereceptorsIntegrins

Selectins

Integrin ligands

Chemokines

Lymphocyte

Figure 3 | A multi-step model for leukocyte entry into the cerebrospinal fluid (CSF). The cartoon shows a portion of the choroid plexus, which is comprised of villi and a centralstroma. The endothelial lumen expresses selectins, such as P-selectin, integrin ligands, such asintercellular adhesion molecule 1 (ICAM1) and immobilized chemokines. A luminal leukocyte,which expresses chemokine receptors, selectin ligands, such as P-selectin glycoprotein ligand 1(PSGL1) and integrins, such as leukocyte function-associated antigen 1 (LFA1) on the cellsurface, is shown sequentially rolling (a), then undergoing engagement of the chemokine receptorwith an immobilized chemokine (b), with subsequent triggering of integrin activation (c), followedby diapedesis (d). Once in the stroma, the leukocyte might be exposed to additional chemokinesthat are produced by epithelial cells of the choroid plexus (e), leading to migration through theepithelial monolayer into the cerebrospinal fluid (CSF) (f). This type of multi-stage migration acrossa vascular endothelium and subsequently across an epithelial barrier has been documented forsubpopulations of B cells120.

Animated online



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five days after adoptive transfer. These protocols favourthe generation of CD4+ T

H1 cells, which have been the

centre of attention for most studies using this diseasemodel. Signs of weakness and incontinence are invari-ably accompanied by inflammatory infiltrates into theCNS, which are composed of T cells and macrophages.

Both active-immunization and adoptive-transferEAE have been used to evaluate mechanisms of accu-mulation of T cells and monocytes in CNS tissues.Despite some progress, the detailed mechanisms bywhich T cells enter inflamed tissues of the CNS remainto be clarified. The first approach to defining lympho-cyte trafficking to tissues of the CNS during EAE was tophenotype infiltrating cells by immunohistochemistryor flow cytometry. The lymphocyte population in early,active-immunization EAE lesions had a phenotype thatwas consistent with newly stimulated memory cellsCD4+CD44+CD45RBlowVLA4+LFA1+ (REF. 41). Followingthese observations, functional studies were carried out,mainly by blockade of these molecules (TABLE 2). As anexample, antibodies specific for α4-integrin reduce theseverity of EAE, even when administered several daysfollowing adoptive transfer42. Such reagents can also beused to treat established EAE, although the effects ofpost-onset administration of α4-integrin-specific anti-bodies were not uniformly beneficial43−45. This approachhas been developed to the point that clinical trials ofα4-integrin-specific antibodies for the treatment of MShave been carried out, with encouraging results46−48.Antibody-induced shedding of CD44 — a hyaluronate-binding protein — expressed by leukocytes also reducedthe severity of EAE without affecting trafficking tolymph nodes49.

It has also been shown that neutralizing antibodiesspecific for LFA1 (integrin αLβ2) can modulate EAE50−53.These results have, however, been thoroughly inconsis-tent. For example, neutralization of the β2-subunit ofLFA1 completely blocked the ability to induce EAE byadoptive transfer in mice, and reduced clinical symp-toms when administrated after the first signs of clinicaldisease53. In other studies, blockade of LFA1 did notsuppress, and even augmented, adoptive-transfer andactive-immunization EAE50−52. In many of these studies,blockade of the targeted integrin supported a role forinteractions at the level of cell extravasation — that is,interactions between the adhesion molecules expressedby leukocytes and the endothelium. However, bothICAM1−LFA1 and VCAM1−VLA4 interactions alsohave important functions during T-cell activation byproviding co-stimulation or adhesive interactionsbetween APCs and T cells. Therefore, the results of indi-vidual experiments must be interpreted in the light ofactivities well beyond trafficking.

Analyses of selectins and their receptors using theEAE model have also not provided simple answers. Insome experiments, expression of selectins was notdetected on the parenchymal endothelial cells of the CNSand antibodies specific for selectin failed to affect diseaseseverity or accumulation of inflammatory leukocytes54,55.In other studies, selectins were detected on cerebrovascu-lar endothelium and blockade of their interactions with

molecules — leukocyte function-associated antigen 1(LFA1)−ICAM1 and α

4β

1−VCAM1 interactions —

could support rolling, as antibody-mediated interfer-ence of either pathway reduced the number of rollingcells. However, blockade of these components did notaffect the rolling characteristics of cells that continued tointeract with the endothelium. This observation wasinterpreted to indicate that integrin–ligand interac-tions were not required for strengthening interactionsduring rolling. However, adhesion did require func-tional interactions between integrin and adhesionmolecules, with LFA1−ICAM1 interactions seeming tohave the main role. The importance of G-protein-coupled receptors for adhesion and arrest was shownby pertussis toxin-mediated abrogation of these events.

Further complexity: don’t forget the spinal cord! All thedata discussed so far concern the brain, yet the spinalcord is the main site of inflammation in EAE, and it isspinal-cord pathology that causes the most severe symp-toms of MS. There are many reasons for considering theinflammatory response of the spinal cord separatelyfrom that of the brain, yet relatively little is known at amolecular level about differences between the two sites.One recent experiment indicated that trafficking eventsin the spinal cord might differ from those that areobserved more rostrally39. In intravital microscopy stud-ies of the dorsal surface of the cervical spinal cord, inves-tigators showed that encephalitogenic T-cell blastsundergo a potentially variant form of tethering at vascu-lar branch points, resulting in the sudden arrest of thecells under flow conditions. This event depends onα4 integrin, but is not sensitive to pertussis toxin39. Oneadvantage of using intravital microscopy to study thespinal cord is that vessels in the central white matterwere visualized directly, owing to the topography of thespinal cord. One overall message to be derived fromthese intravital microscopy analyses of various vascularbeds is that detailed mechanisms of extravasation mightdiffer slightly, depending on the local conditions thatwere studied.

Lymphocyte migration to the CNS in EAEAn initial focus on CD4+ T cells. EAE is a model forexamining the pathogenic mechanisms in demyelinat-ing disorders, of which the most common is the humandisease MS. Investigators generate the model diseaseeither by immunizing animals with myelin components(known as active-immunization EAE) or by transferringencephalitogenic T cells (known as adoptive-transferEAE). Typical active-immunization protocols for rats ormice use defined peptides derived from myelin proteinsas encephalitogenic antigens. Antigens are usually emul-sified in CFA, and injections of B. pertussis toxin are givenat the time of immunization to promote the disruptionof the BBB when inflammation occurs40.Adoptive-trans-fer models involve the generation of encephalitogenic T cells in immunized animals, stimulating cells in vitrowith antigen and cytokines, and injection of these cellsinto naive recipients. Disease begins between two andthree weeks after active immunization, or within about



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C57BL/6 (H−2b) mice were actively immunized withmyelin oligodendroglial glycoprotein (MOG) peptides.Therefore, the contrasting results could have emergedfrom differences in mouse strain or disease model, orbecause gene knockout and antibody-mediated deple-tion did not produce similar physiological effects onchemokine abundance or function during EAE.

Studying the function of CXCR3 in EAE has beenparticularly challenging. In rodents, but not humans,this receptor responds to the lymph-node chemokineCCL21, and potentially mediates the retention of acti-vated T cells in the lymph nodes after priming61.Premature release of these primed cells might explainthe marked worsening of active-immunization EAEthat occurred in rats that received neutralizingCXCL10-specific antibodies62.

From the results of these experiments, it has becomeclear that numerous determinants that are expressed byactivated lymphocytes and used for generic traffickinginto inflammatory sites can be effectively targeted in theEAE model. However, each trafficking determinant thathas been uncovered by these studies has also been docu-mented to function in numerous other inflamed sites.Therefore, the question of whether CNS-specific traf-ficking of determinants are used during EAE remainsunanswered.

A new generation of cell-tracking experiments. Itcould be argued that the application of intravitalmicroscopy and of techniques for labelling and track-ing cells in vivo represents a new wave of usefuldescriptive studies of cell trafficking into the CNS(BOX 2). These studies began more than a decade ago41,with the adoptive transfer of encephalitogenic T cellsthat had been labelled with a lipophilic dye and wereanalysed by flow cytometry. This study also showedthat cells infiltrating the CNS in EAE were activatedmemory T cells, which, at the time of disease onset,were mainly derived from the labelled inoculum, notrecruited from the endogenous T-cell population41.Alternatively, polymorphic-lineage determinants (forexample, Thy1.1/1.2) have been used to track trans-ferred cells63. More recent experiments used cells thathad been engineered to express markers such asgreen-fluorescent protein (GFP) or a clonotypic TCR.In one use of the latter technology, TCR-transgenicmice that were bred onto a recombinase-activatinggene (Rag)-deficient background accumulated naivelymphocytes in tissues of the CNS, in numbers thatwere comparable to the quantities of memory lym-phocytes found in the CNS of non-transgenic Rag-positive mice64. Transgenic T cells that are specific foreither a neuroantigen, such as myelin-basic protein(MBP), or an irrelevant antigen (ovalbumin) werefound in equivalent numbers in the CNS of thesemice, showing conclusively that antigen specificity hasno role in the accumulation of T cells in the CNS.Importantly, these results indicated that requirementsfor lymphocytes to enter the tissues of the CNS arenot coincident with functions that are expressed byactivated or memory cells64.

leukocytes affected trafficking36,38. The fact that differ-ing techniques, disease models and mouse strains wereused in the various studies might account for the dis-crepancies. The most recent data support a role for P-selectin in rolling on inflamed cerebrovascularendothelium36,38,56. A further twist to the selectin−EAEstory reminds investigators that molecules have func-tions beyond those that are initially described for them.L-selectin-knockout mice failed to develop EAE, evenafter being crossed to myelin-antigen-specific TCR-transgenic animals57. This protective effect on EAE was,however, not due to reduced infiltration of leukocytesinto the CNS, as further analysis showed that expres-sion of L-selectin by macrophages was required formediating the effector function and final destruction ofCNS myelin57.

The chemokines and chemokine receptors have alsobeen difficult to assign specific lymphocyte-recruitmentfunctions during EAE. Neutralizing antibodies specificfor CC-chemokine ligand 3 (CCL3) or CXCL10 pro-tected against disease, most markedly when deliveredafter adoptive transfer58,59. However, both CCL3- andCXCL10-knockout mice were entirely susceptible toEAE, leaving the specific functions of these chemokinesin this disease model unresolved (A. Luster, personalcommunication)60. Further complicating the situation,antibody-mediated blockade of chemokines was carriedout in SJL (H−2s) mice, using a passive EAE model inwhich recipients were transferred with T cells that wereresponsive to a proteolipid protein (PLP) peptide,whereas for the study of chemokine-knockout mice,

Box 2 | Approaches to the study of leukocyte trafficking to the CNS

In small animal systems• Injections of leukocytes labelled ex vivo

• Intrathecal inoculation of antigen

• Administration of antibodies against trafficking determinants

• Transgenic expression of trafficking determinants under the control of tissue-specificpromoters

• Knockout animals

• Experimental models of inflammation of the central nervous system (CNS) in animals(for example, experimental autoimmune encephalomyelitis)

• Stamper−Woodruff assays that measure leukocyte adhesion to sections of frozen brain

• Intravital microscopy of CNS microvasculature

In humans• Descriptive analysis of chemokines in body fluids — peripheral blood and

cerebrospinal fluid (CSF) — and chemokine receptors expressed by circulating cells in these compartments

• Functional in vitro analysis of the trafficking capacity of cells of the CSF

• Expression of trafficking determinants by resident and infiltrating cells in CNSparenchyma (autopsy and biopsy tissue)

• Stamper−Woodruff assays that measure leukocyte adhesion to sections of frozen brain(autopsy and biopsy tissue)

• Solution-phase functional-adhesion assays using, for example, selectin−immunoglobulin fusion-protein constructs to assay activated forms of selectin-binding glycoproteins or cell-adhesion molecule (CAM)−immunoglobulin fusionconstructs to assay activated integrins



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during their residence in the spleen65. It is plausible that,while in the spleen, these highly-activated cells mediatethe release of cytokines that activate the endothelium —by analogy to intravenous injections of TNF or LPS.Further studies that use labelled encephalitogenic T cellsmight uncover selective regulators of T-cell trafficking tothe CNS. One unexplored question is: what are the rele-vant sites of initial trafficking during the acceleratedphase of migration that characterize the onset of EAE? Inparticular, do cells first accumulate in the CSF or in themeningeal sites, and subsequently enter the parenchymaor traverse to the parenchymal vessels directly?

What about other leukocyte cell types?CD8+ T cells. So far, the main focus of EAE studies hasbeen CD4+ T cells. Recent results indicate that CD8+

T cells can mediate EAE and they are the main popula-tion that is clonally expanded in the brain of individualswith MS68,69. Furthermore, CD8+ T cells distributewidely throughout the parenchyma in inflammatorydisorders as varied as MS, Rasmussen’s encephalitis andthe paraneoplastic encephalitides70. Little is knownabout trafficking of CD8+ T cells into the CNS; however,the differential distribution patterns of CD4+ and CD8+

T cells in inflammatory lesions of CNS indicate that differences between these populations will be marked.

Natural killer cells. During the inflammatory process thatoccurs during adoptive-transfer EAE, natural killer (NK)cells are among the earliest recruits34,37. Expression ofchemokine receptors and chemokine responsiveness ofNK cells differs markedly from those characterized for T cells71. Therefore, it is possible that selective-traffickingdeterminants will emerge for NK cells as comparedwith other lymphocyte populations.

γδT cells. γδT cells constitute a distinctive lymphocytepopulation that preferentially localizes to epithelial sur-faces. Their role in EAE is somewhat controversial butseems to be largely modulatory72,73. Notably, γδT cellswere the main population that infiltrated the CNS ofmice infected with Mesocestoides corti — an animalmodel of neurocysticercosis74,75. Although traffickingdeterminants of this population remain to be com-pletely defined, M. corti-infected CCR5-knockout miceshowed a reduction in γδ T cells in the meningeal com-partment of the CNS, correlating with an increasedparasite burden (J. Teale and A. Cardona, personalcommunication).

Mononuclear phagocytes. Mononuclear phagocytes arethe main components of leukocytes in most CNSinflammatory reactions. These cells are represented inthe circulation by monocytes, and study of the traffick-ing of monocytes to the CNS is the appropriate focalpoint for understanding how this lineage enters sites ofinflammation in the CNS. Their relevance is confirmedby experiments showing that depletion of monocytesessentially abrogates the development of EAE76,77.Because they function mainly as effectors of inflamma-tory reactions, it is unlikely that monocytes enter healthy,

Studies in rats that received GFP-labelled encephalito-genic T cells provided strong evidence that favours theexistence of a specific post-transfer programme of cellactivation that leads to the accumulation of lymphocytesin the CNS65. Following antigen stimulation in vitro,encephalitogenic T cells expressed high levels of activationantigens, such as OX40, and low levels of chemokinereceptors. After transfer, GFP-labelled MBP-specific lymphocytes rapidly moved from the parathymic lym-phoid tissue through the blood and accumulated in the spleen, where they downmodulated the expression ofactivation markers and upregulated the expression ofchemokine receptors, including homeostatic receptorssuch as CCR7, CXCR4 and several inflammation-relatedCC-chemokine receptors. Remarkably, there followed anear-synchronous relocation of encephalitogenic cellsfrom the spleen to the CNS at the onset of disease, wheremigration-associated gene expression was suppressed andactivation markers were re-expressed65.When encephali-togenic T cells were replaced by identically labelled andpre-activated ovalbumin-specific cells, early migrationpatterns were similar, but subsequent patterns of marker regulation in the CNS (as well as cell persistence and dis-ease) failed to occur65. This result indicated a requirementfor local re-stimulation with antigen for the preciseregulation of expression of activation and migrationdeterminants in the encephalitogenic T-cell population.

Early events after the transfer of activated neuro-antigen-specific T cells have also been re-analysed,through the use of labelled cells and high-resolutionmicroscopy. It was shown more than a decade ago that afew activated cells accumulate in the CNS ~6 hours aftersuch transfers66. The recent studies were focused at 2 hours after transfer, and showed clearly that these earli-est CNS immigrants accumulate in the choroid plexusor meninges24,67. Furthermore, reagents that block theinteraction of P-selectin with its carbohydrate ligandsmarkedly reduced this trafficking, whereas neutral-ization of VLA4−VCAM1 interactions had no effect24,67.Additional studies showed expression of P-selectin in thechoroid plexus and meninges of healthy mice, indicatingits function as a trafficking determinant for entry intothis site24.

Placing the presently available information in con-text, it seems that adoptive transfer of primed encepha-litogenic CD4+ T cells leads to the accumulation of asmall number of cells in the meninges, the choroidplexus and, perhaps, the parenchyma, within 2 hoursafter transfer. P-selectin is likely to be implicated in thisearly trafficking. This low-efficiency first wave of cellmigration might function to activate parenchymal ves-sels for interactions with the subsequent large-scale entryof leukocytes, as the most recent intravital microscopydata indicate that non-activated parenchymal vessels arerefractory to communication with lymphocytes, eventhough they are primed and neuroantigen-specific38.However, the physiological relevance of this early cellmigration remains to be established.

Most CD4+ T cells, after transfer, undergo a highlystructured migratory programme, with a phase ofregulated expression of functional cell-surface markers



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CNS, as observed in cases of MS, subacute sclerosingpanencephalitis (SSPE) and neurosyphilis. There isvirtually nothing known about mechanisms of B-cellmigration in the setting of CNS inflammation, althoughelegant experiments using intraparenchymal instilla-tion of antigen showed that B cells can accumulate in tissues of the CNS without previous disruption ofthe BBB94.

Neutrophils. Neutrophils enter CNS tissues only underextreme circumstances21. Expression of CXCR2 ligands,such as CXCL1, either by transgenic mice or delivered byrecombinant adenoviruses, can force invasion of neu-trophils into the CNS parenchyma95−97. A bacterialabscess provides a well-characterized model for whichneutrophils are pivotal elements in host defence98.Expression of CXCR2 ligands is elevated in the CNSof mice during early abscess formation and CXCR2-deficient mice have reduced neutrophil infiltrationwith worse outcomes98,99.

Unresolved issues and future directionsThe past decade has seen prodigious increases in ourknowledge about leukocyte trafficking. There is also asubstantial amount of experimental data on the accu-mulation of leukocytes in the CNS. It is now importantto interpret these data in such a way as to assemble acollection of meaningful and testable hypotheses aboutthe mechanisms of leukocyte entry into CNS tissues.Experiments that selectively examine cell traffickinginto meningeal, ventricular or parenchymal sites will beextremely useful, and have already produced valuableinsights. We are doubly challenged, first, by the com-plexity of the vascular anatomy of the CNS and, second,by the many functions of molecules that are understudy, many of which have properties well beyond thoseinvolved in trafficking100. The loss of these activities afterantibody-mediated blockade or gene deletion oftenproduce controversial results, requiring ingeniousstrategies to test trafficking functions selectively.

Note added in proofWhile this review was in press, it has been shown byKivisäkk and colleagues121 that human central memoryCD4+ T cells traffic into the CSF across either thechoroid plexus or the meningeal vessels. In addition,they show that this trafficking is mediated through P-selectin that is expressed either by the veins of thechoroids plexus or vessels within the meninges.

unactivated tissues of the CNS. Therefore, the examina-tion of monocyte trafficking to the nervous systemfocuses, mostly, on the response to ongoing inflamma-tion. Mice that lacked either CCR2 or one of its ligands,CCL2, were relatively resistant to the development ofEAE, despite having T-cell responses that were equiva-lent to those observed in wild-type mice78−80. The CNStissues of CCL2-deficient mice were virtually devoid ofmonocytes at time points equivalent to those at whichwild-type mice developed neurological impairment79.CCR2 was also essential for the accumulation of mono-cytes at the epicentre of a spinal-cord contusion, andtheir absence in CCR2-deficient mice was associatedwith impaired clearance of tissue debris81. Mice thatlack CCR1 also had milder EAE than controls, withreduced accumulation of monocytes in affected CNStissues82. It is uncertain what specific roles each ofthese receptors have in the processes of monocytemigration to the inflamed CNS. Detailed examinationof the complementary roles of CXCR2 and CCR2 inmodels of atherosclerosis indicated a division of labour,which assigned to CXCR2 the function of arrestingmonocytes under flow, whereas CCR2 mediatedtransendothelial migration83,84.

Dendritic cells (DCs) are the professional APCs formost immune reactions85. Whereas no DCs have beendetected in the intact brain parenchyma, such cells arepresent in the meninges and choroid plexus of healthyrodents86. Recently, it has become increasingly clearthat cells expressing DC markers are present in thebrain during several inflammatory conditions, includ-ing ischaemic stroke, cerebral toxoplasmosis andEAE87−90. DCs can be derived from monocytes or froma lymphoid precursor. Both myeloid and lymphoidDCs have been detected in the CSF of humans withinflammatory conditions, usually acute or chronicmeningitis91,92. These DCs express CCR5, a chemokinereceptor that is expressed by immature DCs93. By con-trast, myelin antigen-containing DCs that are detectedin cervical lymph nodes of EAE in primates wereCCR7+, indicating a mature phenotype11. Together,these results suggest that DCs can migrate between theCNS and the periphery in neuroinflammation. Theexact migration or differentiation patterns of DCs inthe CNS remain, however, a matter of conjecture.

B cells. B-cell migration to the CNS is a poorly under-stood phenomenon. It is well established that intrathecal-antibody synthesis occurs during inflammation of the

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AcknowledgementsThe authors would like to thank S. M. Staugaitis and J. J. Campbellfor an insightful critique of this paper. We also acknowledge J. Teale, A. Cardona and A. Luster for communicating resultsbefore publication. Research in Dr. Ransohoff’s laboratory is supported by the National Institutes of Health.

Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/CCL21 | CCR1 | CCR2 | CCR3 | CCR5 | CCR6 | CCR7 | CD4 |CD8 | CD44 | CXCL10 | CXCR2 | CXCR3 | CXCR4 | ICAM1 | MBP | OX40 | PECAM | P-selectin | PSGL1 | TNF | VCAM1 | VLA4OMIM: http://www.ncbi.nlm.nih.gov/Omim/Alzheimer disease | multiple sclerosis

FURTHER INFORMATIONWorkshop on choroid plexus: http://www.lyon.inserm.fr/choroid_plexus/G-protein-coupled receptors datatbase: http://www.gpcr.orgChemokine database: http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.htmlAccess to this interactive links box is free online.

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