The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms

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The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms Britta Engelhardt1 and Richard M. Ransohoff2 1

Theodor Kocher Institute, University of Bern, Freiestr. 1, CH-3012 Bern, Switzerland The Mellen Center for Multiple Sclerosis Treatment and Research, and Department of Neurosciences of The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, NC-30, Cleveland, OH 44195, USA 2

This review addresses current knowledge of the molecular trafficking signals involved in the migration of circulating leukocytes across the highly specialized blood–central nervous system (CNS) barriers during immunosurveillance and inflammation. In this regard, adhesion molecules and activating and chemotactic factors are also discussed and the regional variability in the brain and spinal cord parenchyma are also considered. Furthermore, direct passage into cerebrospinal fluid (CSF) is discussed, in the context of CNS immunosurveillance. The potential differences that characterize leukocyte entry into these varied anatomical sites are highlighted, with special emphasis on studies of the pathogenesis of multiple sclerosis and its animal models. An update on findings from clinical trials of natalizumab is also provided.

Introduction and background Central nervous system (CNS) homeostasis is crucial for the proper function of neuronal cells. The seminal observation of Sir Peter Medawar, that the brain does not reject foreign tissue grafts [1], was followed by complementary findings: the CNS is devoid of classical antigen-presenting cells (APCs), such as dendritic cells (DCs); the CNS lacks constitutive MHC I and II expression on parenchymal cells; lymphatic vessels are not present in the CNS. Together, these data led to the conception that the CNS represents an immune privileged site. Because the CNS is additionally protected from the continuously changing milieu of the periphery by the endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid (CSF) barrier (BCSFB), the term ‘immune privilege’ was interpreted to indicate a complete absence of immunosurveillance within the CNS. There is now agreement that this view is too extreme: CNS tissue grafts are rejected eventually; delayed type hypersensitivity reactions occur in the CNS; and pathogenic CNS autoimmunity is exemplified by experimental autoimmune encephalomyelitis (EAE). Given the delicate network of post-mitotic, non-renewing neural cells that Corresponding authors: Engelhardt, B. ([email protected]), Ransohoff, R.M. ([email protected]). Available online 21 July 2005

populate the CNS and the catastrophic consequences of intrathecal swelling, CNS inflammatory reactions are modified. It is more appropriate to consider the CNS to be immune specialized, and acknowledge that immunosurveillance of the CNS is a crucial component of host defence. Further, initiation points for pathogenic inflammatory reactions in the CNS must exist. In this review, how the trafficking of lymphocytes through the CNS supports these processes is discussed. During a variety of pathological conditions in the CNS, such as viral infections, ischemia or inflammatory diseases [such as multiple sclerosis (MS)], leukocytes traverse the BBB and accumulate within enlarged perivascular spaces, following which many cells find their way into the CNS parenchyma. Although EAE infiltrates are classically devoid of neutrophils, careful observation discloses a minority population of neutrophils in some EAE models [2–4]. By contrast, bacterial infection of the meningeal spaces preferentially induces a massive influx of neutrophilic granulocytes into the CSF. Thus, depending on the inflammatory stimulus and the CNS compartment affected, molecular pathways for leukocyte entry into the CNS are different. There is an extensive body of knowledge about the molecular trafficking signals involved in the migration of neuroantigen-specific autoaggressive T lymphocytes, which enter the CNS during EAE. Peripheral injection of encephalitogenic lymphocytes into a syngeneic healthy host or subcutaneous immunization with myelin antigens in complete Freund’s adjuvant elicits EAE, an inflammatory demyelinating and necrotizing disease of the CNS, which bears points of similarity to the spontaneous human disorder, MS. During the course of EAE, autoaggressive CD4C T lymphocytes are activated outside the CNS and accumulate behind the blood–CNS barriers, where they initiate the cellular events leading to inflammatory tissue destruction. Therefore, EAE presents an excellent model to study leukocyte migration across blood–CNS barriers during autoimmune inflammation. Many of the considerations summarized in this review will be based on observations made in EAE and in MS, leaving open the strong possibility that other mechanisms apply to inflammatory cell recruitment into the CNS under different pathological conditions.

www.sciencedirect.com 1471-4906/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2005.07.004

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Anatomical routes for circulating leukocytes to cross blood–CNS barriers The endothelial BBB has been considered the most obvious point of entry for circulating immune cells into the CNS (Figure 1). The term BBB originally described the lack of passive diffusion of molecules across the CNS capillaries. The BBB is formed by highly specialized endothelial cells, which inhibit transcellular molecular traffic owing to low pinocytotic activity, and restrict paracellular diffusion of hydrophilic molecules because of an elaborate network of complex interendothelial tight junctions [5]. Leukocyte extravasation occurs solely at the level of post-capillary venules and thus we suggest that the term BBB should encompass both capillaries and postcapillary venules, given equally restricted diffusion of polar molecules. No differential characteristics of CNS post-capillary venules within brain and the spinal cord, or the CNS grey versus white matter have been reported. However, there are subtle differences between the BBB characteristics of meningeal and parenchymal microvessels [6] because the former but not the latter lack astrocytic ensheathment, whereas the latter are deficient for P-selectin storage [7,8].

Fenestrations and intercellular gaps [9] permit free movement of molecules across the microvessels of the choroid plexus and circumventricular organs (CVOs). The BCSFB of the choroid plexus is comprised of tight junctions between choroid plexus epithelial cells. Trafficking of T lymphocytes through the non-inflamed CNS and routine immunosurveillance CNS inflammatory reactions (both pathologically and in the service of host defence) implicate mechanisms for immunosurveillance. Physiological trafficking of lymphocytes through the CNS supports the essential function of immunosurveillance; however, the study of lymphocyte entry into the non-inflamed CNS has not provided unambiguous results, probably owing to variation in the experimental approach. Interaction of T lymphoblasts with brain parenchymal vessels The first direct evidence that lymphocytes enter the CNS in the absence of overt inflammatory disease [10,11] came from tracing radioactively labelled encephalitogenic T-cell blasts following their intravenous injection into Lewis (a)

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Figure 1. Afferent and efferent CNS immune pathways. The different CNS compartments, choroid plexus, cerebral ventricle, brain, spinal cord parenchyma and subarachnoid space are shown, together with their possible connections to the peripheral immune system (blood vessels and lymphatics of the nasal mucosa). Efferent connections from the immune system to the CNS take various forms, including entry of circulating T-cell blasts across the BBB into the brain, across the blood–spinal cord barrier (BSpCB) into the spinal cord parenchyma and across the BCSFB (established by the choroid plexus epithelium) into the CSF-filled ventricles. APCs of the CNS are depicted in yellow and are located in the choroid plexus stroma ‘in front’ of the barrier [choroid plexus macrophages (CPMs)], subarachnoid spaces [meningeal macrophages (MMs)], the perivascular or Virchow-Robin spaces [perivascular cells (PVCs)] or on the apical surface of choroid plexus epithelium (Kolmer cells). Interstitial CNS fluid drains into the perivascular spaces around arteries and alongside the nasal olfactory artery to the cribriform plate. From there it enters channels in the arachnoid, which are continuous with the lymphatics in the nasal mucosa, draining into the regional cervical lymph nodes By this route, afferent connections of the immune system to the CNS have been established to the regional cervical lymph nodes. Barriers are depicted in red; notably, ependymal cells lining the ventricles do not form a barrier. (a) Depicted in greater detail in Figure 2a. (b) Depicted in greater detail in Figure 2b. (c) Depicted in greater detail in Figure 2c. www.sciencedirect.com

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rats. Resting lymphocytes fail to enter the CNS [11], suggesting that only activated T lymphocytes penetrate the BBB under these conditions. Activation-stage, rather than the antigen-specificity of CD4C T lymphoblasts, determines BBB crossing in this model [10]. Beyond a few hours, however, neuroantigen specificity is needed for lymphoblast persistence in the CNS. These T lymphoblasts reside in the CNS perivascular spaces [12], permitting encounter with antigen presented by MHC II-positive perivascular macrophages. Monitored 6 hours post-injection, T lymphoblasts enter the CNS at extremely low efficiency, with w0.01 detected cells per gram of tissue per 100 injected cells, which is 100-fold less than in spleen or lung [13]. Persistence of neuroantigen-specific T lymphoblasts, with their perivascular localization, suggests an immunological function for these cells. However, not all studies provided identical results. By 2 hours after peripheral injection, fluorescently labelled T lymphocytes can be readily detected in the meninges and the choroid plexus but not in the brain parenchyma [14]. Intravital microscopy (IVM) studies through the intact skulls of young mice failed to detect the interaction of autoaggressive T-cell blasts with non-inflamed superficial brain microvasculature [15], suggesting that the infrequent T-lymphocyte migration into meninges reported by others [14] might be missed by this approach. Activation of microvessels by earlier injection of lipopolysaccharide (LPS) or tumour necrosis factor-a (TNF-a) enables the efficient interaction of T lymphoblasts with superficial cerebral vasculature [15], suggesting that systemic stimuli could prime the CNS for inflammation.

Extravasation of T lymphoblasts across spinal cord parenchymal vessels Interaction of encephalitogenic T lymphoblasts with the microvasculature in the non-inflamed spinal cord white matter can be detected readily by IVM [16] (Box 1). Uniquely, encephalitogenic T-cell blasts fail to roll and are instead captured promptly within the spinal cord white matter microvessels (Figure 2b), a process dependent on a4 integrins and vascular cell adhesion molecule-1

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(VCAM-1). A G-protein-mediated increase in integrin avidity on the T-lymphocyte surface in situ, which is necessary for adhesion strengthening dependent on a4 integrins and VCAM-1, follows [16]. Leukocyte functionassociated molecule-1 (LFA-1) supports the migration of these T lymphocytes across the spinal cord white matter microvascular wall, which seems to follow specialized mechanisms because diapedesis of T lymphocytes is only observed 3 hours post-injection [17], compared to minutes in other vascular beds [18].

Interaction of T lymphoblasts with retinal microvessels These somewhat disparate findings in brain and spinal cord can perhaps be integrated and clarified by recent studies of the blood–retina barrier (BRB) [19], in which microvessels exhibit tight junctions and barrier characteristics identical to those found in the CNS parenchyma. To study the BRB, IVM was combined with whole-mount analysis of total migrated fluorescent cells. Activated T lymphoblasts cross the BRB at approximately the same rate as shown before for spinal cord: 0.01 cells per gram of tissue per 100 injected cells. Only activated cells cross and do so by an immediate-arrest mechanism closely reminiscent of that described previously for spinal cord. Pretreatment of transferred cells with anti-LFA-1 antibodies suppresses infiltration, however, the relative contributions of LFA-1 and a4-integrins to capture, compared with diapedesis, were not addressed. T lymphoblasts fail to cross retinal venules until 8 hours post-injection, by which time modest BRB disruption (Evans blue–albumin complex extravasation) and endothelial intercellular adhesion molecule-1 (ICAM-1) upregulation had already occurred. Based on results from numerous laboratories, T-lymphoblast extravasation across BRB and BBB after intravenous injection exhibits the following characteristics: (i) the procedure of T-lymphoblast injection mediates microvascular endothelial cell activation and modulates barrier function, establishing pre-conditions for capture and extravasation; (ii) T lymphoblasts expressing high levels of activated integrins but not nonactivated lymphocytes cross this ‘pre-conditioned’ CNS

Box 1. The multi-step process of leukocyte extravasation The recruitment of leukocytes from the blood into different tissues is a tightly controlled process that directs the extravasation of the appropriate cell to the proper location at the right time, thus ensuring immunosurveillance of the body. Leukocyte migration across the vascular wall is a multi-step process [92–-94]. The multi-step paradigm postulates that an initial transient contact of the circulating leukocyte with the vascular endothelium, generally mediated by adhesion molecules of the selectin family and their respective carbohydrate ligands, slows down the leukocyte in the bloodstream. Uniquely, lymphocytes can also roll via the interaction of a4-integrins with their endothelial ligands VCAM-1 or MAdCAM-1. After an initial tethering, the leukocyte rolls along the vascular wall with greatly reduced velocity. The rolling leukocyte can now ‘sense’ chemotactic factors from the family of chemokines, which are presented on the endothelial surface and which bind to their cognate serpentine receptors on the leukocyte. These receptors deliver a G-protein-mediated pertussis toxin-sensitive signal into the leukocyte, resulting in the functional activation of adhesion molecules of the integrin family on the leukocyte surface. Activation of integrins leads to an increase in their www.sciencedirect.com

affinity and/or avidity. Only ‘activated’ integrins are able to mediate the firm adhesion of leukocytes to the vascular endothelium by binding to their endothelial ligands from the Ig-superfamily of adhesion receptors. This process ultimately leads to leukocyte diapedesis, through inter-endothelial cell junctions or directly through the endothelial cell itself. Diapedesis involves integrins and molecules of the endothelial adherens junctions and (where present, such as in the BBB) tight junctions. Successful recruitment of circulating leukocytes into the tissue depends on a productive leukocyte–endothelial interaction during each of these sequential steps. Tissue-specific display of adhesion molecules and chemokines by vascular endothelium and the selective expression pattern of their ligands on leukocyte subsets provides a combinatorial mechanism for specificity and diversity in targeting different leukocyte subpopulations to different organs. The molecular sequence of traffic signals involved in T-lymphocyte homing to the lymph nodes during immunosurveillance, as well as T-lymphocyte migration to the body surfaces (i.e. the gut and the inflamed skin), have been established using antibodyinhibition studies, knockout mice and IVM [93].

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microvasculature; (iii) extravasation is relatively slow (O3 hours post-injection), exhibits low efficiency and often starts with immediate arrest, which does not require selectins; (iv) endothelial cell activation and compromised barrier function might be induced either by lymphoblast– endothelial contact, which initiates LFA-1-dependent signalling to the ICAM-bearing endothelial cell, or by secreted proinflammatory lymphoblast products. T lymphocytes in the CSF Under physiological conditions, the CNS strictly controls immune cell entry across its barriers and localizes its immunosurveillance to the perivascular and subarachnoid spaces, leaving the CNS parenchyma untouched. The CSF of healthy individuals contains w150 000 cells, O80% of which are CD4CCD45ROCCD27CCXCR3C central-memory T lymphocytes (retaining lymph node homing determinants CCR7C and L-selectinhi), about half of which are CD69C, indicating that such cells routinely penetrate the choroid plexus epithelium. Furthermore, the cells re-enter the bloodstream and are replaced by newly immigrating lymphocytes approximately twice per day, providing a continuous flux through the CNS of lymphocytes with differing specificities. These CSF T lymphocytes are enriched greatly compared with circulating blood, in which they constitute !5% of leukocytes. Surprisingly, P-selectin is involved in the entry of murine T lymphoblasts into the meninges and choroid plexus because antibodies blocking P-selectin or injection of T lymphocytes into P-selectin-deficient mice reduced significantly the number of T lymphocytes found within these compartments [14], suggesting that endothelial cells in the meninges and the choroid plexus express constitutively low levels of surface P-selectin, below detection by immunohistochemistry (IHC) [20,21] (Figure 2c). Consistent with this proposal, immunohistochemical evaluation of the control brains, obtained during autopsy of individuals who died without neurological disease, uniformly reveals P-selectin immunoreactivity at two locations associated with trafficking into the CSF: the deep stromal veins of the choroid plexus and the bridging meningeal veins [22]. By contrast, P-selectin immunoreactivity is not found in the corresponding murine structures [20,21]. T-lymphoblast immigration across parenchymal vessels is not observed in short-term migration studies [14], possibly related to the time required for endothelial activation but also concordant with previous observations that brain parenchymal endothelium lacks storage of P-selectin in

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Weibl-Palade bodies [7] and recent findings that the parenchymal CNS vasculature of humans lacks P-selectin, even in MS lesions [22]. Detection of adhesion molecules by IHC is often difficult and either positive or negative results should be regarded as preliminary. Positive results require confirmation both by complementary biochemical means (such as ISH) and by functional analysis using neutralizing antibodies, small-molecule inhibitors (in either the pre-clinical or clinical setting) or knockout mice. However, negative IHC results should also be considered preliminary because of the low level of adhesion molecule expression by cerebrovascular endothelium and the need for consistent findings from multiple laboratories and the varied antibody preparations to be regarded as definitive. One study argued against a role for a4-integrin in T-lymphocyte traffic into the non-inflamed brain because the anti-a4-integrin antibody R1.2 failed to affect T-lymphocyte migration in meninges [14]. However, because R1.2 inhibits a4-integrin-mediated lymphocyte migration poorly in vivo [23,24], this issue remains to be addressed definitively. Although mechanisms of migration are not fully defined, cumulative data suggest that lymphocytes enter the CSF across the choroid plexus during immunosurveillance. In further support of this concept, the cellular composition of ventricular and lumbar CSF in patients without CNS inflammation is identical and composed mainly of CD4C central-memory T lymphocytes [25]. Immunosurveillance of the CNS parenchyma It has been proposed that CSF central-memory CD4C T lymphocytes carry out routine immunosurveillance of the CNS, by searching within the CSF-filled subarachnoid spaces for recall antigens presented by either the rich network of subarachnoid-space macrophages, many with DC properties [26], or by pericytes embedded in the basal membranes of the CNS microvasculature (Figure 1). Under most circumstances, the initial encounter of naı¨ve lymphocytes with neuroantigen occurs in deep cervical lymph nodes. By corollary, this concept pre-supposes efficient drainage of CNS antigens from parenchyma to the CSF circulatory pathways, either over the convexities or within the ventricles, being carried finally to the deep cervical lymph nodes [27]. Further, a small population of CCR7C mature DCs can be detected in human CSF. Thus, despite the lack of classical lymphatic vessels, efferent and afferent connections of the immune system and the CNS

Figure 2. Multi-step recruitment of T lymphocytes across the blood–CNS barriers. An overview of the adhesion and signaling steps involved in T-lymphocyte migration across the blood–CNS barriers, based on expression patterns of the relevant molecules and IVM, is shown. (a) In the inflamed brain, E- and-P-selectin, their leukocyte ligand PSGL-1 and a4-integrin are involved in lymphocyte tethering and rolling in superficial brain vessels. G-protein-dependent activation of LFA-1 on T lymphocytes leads to their firm adhesion on endothelial ICAM-1. Lymphocytes migrate transcellularly through the BBB endothelium, leaving tight junctions morphologically intact. In EAE, the inflammatory lymphocytes present in brain (and spinal cord) parenchyma are Th1 effector memory cells with a characteristic CD45RBlowICAM-1highLFA-1highCD44higha4b1higha4b7low/K L-selectinlow/K surface phenotype. (b) In the non-inflamed spinal cord white matter, T lymphocytes do not roll, but arrest through a4-integrin on endothelial VCAM-1. G-protein-dependent increase in a4-integrin avidity on the T lymphocytes is required for firm adhesion to endothelial VCAM-1. CCL19 is expressed by spinal cord microvascular endothelial cells. LFA-1 supports T-lymphocyte diapedesis, adjacent to tight junctions. During EAE, upregulated expression of endothelial CCL21 and ICAM-1 might lead to their involvement in this process. (c) Less is known about molecular mechanisms in lymphocyte transit across the fenestrated choroid plexus microvessels and subsequent migration across the choroid plexus epithelium into the CSF. In the non-inflamed CNS, endothelial P-selectin mediates T-lymphocyte recruitment into the choroid plexus stroma. Murine choroid plexus epithelial, but not endothelial, cells express ICAM-1 and VCAM-1, which are upregulated under inflammatory conditions, during which de novo expression of MAdCAM-1 is also observed. CCL21 has been detected in the choroid plexus, implying a possible role in lymphocyte recruitment into the CSF. Pathways of T-lymphocyte traversal across choroid plexus endothelial and epithelial barriers are equally mysterious. T lymphocytes in the CSF of healthy individuals are mostly central memory CD4C T lymphocytes. www.sciencedirect.com

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are relatively robust. In contrast to previous findings [28,29], recent results underline that non-activated T lymphocytes can enter the CNS parenchyma during EAE, and can be activated in situ by cognate antigen [30]. Therefore, one can anticipate that the mechanisms of physiological immunosurveillance are distinct from those operating during disease. The role of lymphoblast extravasation in routine CNS immunosurveillance is uncertain. It seems unlikely that this mechanism alone accounts for such a crucial function because the number of extravasated cells per day would be small (maximally 105 for the entire CNS) given that only a minimal population of activated lymphoblasts are present in circulating blood (no more than 1 in 104 lymphocytes). Furthermore, it seems inconceivable that isolated, individual extravasated T lymphocytes would encounter cognate antigen in the confined perivascular spaces of parenchymal vessels containing solitary perivascular DCs [31]. Finally, activated lymphoblast cells downregulate CCR7 and L-selectin, depriving them of lymphoid homing signals. By contrast, it seems plausible that this type of encounter represents an initiating event for the inflammatory cascade in adoptive-transfer EAE, in which large numbers of cells are injected and in which there is a reasonable likelihood that highly abundant self-antigens might be presented by perivascular DCs [30–32]. It is pertinent that there are two phases of autoaggressive T-cell accumulation in the CNS following adoptive transfer: (i) several hours post-transfer, a small population of potential ‘initiator’ cells are observed in the CNS parenchyma and meninges; (ii) the vast majority of cells re-circulate through lymphoid organs, upregulate chemokine receptors and acquire efficient migratory capacity, and accumulate with enormous rapidity in the CNS, 72 hours after peripheral injection [33]. Recent results indicate that the peripheral lymphoid tissue is dispensable for T lymphocyte-dependent neuroinflammatory disease [31] but confirm that perivascular DCs [30] are responsible for re-activation and retention of neuroantigen-specific T lymphocytes in the CNS. These cumulative observations strongly support the concept that leukocyte trafficking mechanisms during immunosurveillance and disease are distinct.

Immigration of T lymphocytes across the blood-CNS barriers during inflammation: molecular mechanisms In inflammatory conditions of the CNS, the expression of adhesion molecules and chemokines is induced on BBB endothelium and the choroid plexus epithelium, providing additional traffic signals for circulating leukocytes (Box 1). Thus, a significant number of leukocytes enter the CNS, making this process easier to investigate. Interestingly, even when the endothelial BBB becomes leaky, T-lymphocyte recruitment into the CNS remains tightly controlled because parenchymal lymphocytes comprise a homogeneous memory/effector phenotype with a unique adhesion molecule display and are thus phenotypically distinct from T-lymphocyte populations in other inflamed organs [28,29,34] (Figure 2a). www.sciencedirect.com

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Tethering and rolling of leukocytes on inflamed BBB endothelium During EAE, or following intravenous TNF-a, tethering and rolling of blood leukocytes can be observed readily in superficial brain and meningeal microvessels by performing IVM using a cranial window [35] or through the intact skulls of young mice [36]. In inflamed murine vessels, CD8C T lymphocytes from MS patients preferentially roll via P-selectin glycoprotein ligand-1 (PSGL-1), whereas CD4C T lymphocytes roll via a4-integrin [36]. By contrast, PSGL-1–E- or P-selectin interactions are implicated in Th1-cell recruitment into inflamed retina [37]. During EAE, both a4-integrin- and PSGL-1-mediated leukocyte rolling occur in brain vessels [35]. Besides P-selectin, E-selectin can mediate leukocyte rolling in inflamed superficial brain vessels [15,38]. Accordingly, it is surprising that E- and P-selectin antibodies do not influence the development of EAE in SJL mice [20] and blocking PSGL-1 or the lack of PSGL-1 fails to affect adoptively transferred or actively induced EAE [39]. Analyzing the migration of regulatory lymphocytes or studying the roles of adhesion molecules (including PSGL-1) in regulatory cell–cell interactions might be required to resolve this conundrum. Inflammation in EAE is mainly localized to the spinal cord white matter. As noted earlier, leukocyte recruitment to the spinal cord white matter might not rely on selectin-mediated rolling (Figure 2b). Consistent with this view, L-selectin is not involved in EAE [40,41], whereas a4-integrin and CD44 might have crucial roles in this disease [40].

Activation step Integrin activation, mediated by signalling through G-protein-coupled receptors, is essential for leukocyte arrest under flow conditions. Chemokines and their receptors are the largest family of molecules that deliver such signals and they also orchestrate temporo-spatial cellular localization in developmental organ patterning and immunity. IVM studies of T-lymphocyte interactions with the superficial brain and spinal cord microvasculature demonstrate that G-protein signalling is required for the firm arrest of autoaggressive T lymphocytes within the CNS microvessels [15,16]. For extravasation of lymphocytes into the CNS, relevant questions are: (i) which chemokines (or other Gi-linked signals, such as eicosanoids) are present and what is their function; (ii) which receptors are expressed on target lymphocytes; and (iii) how are chemokines transcytosed from the abluminal parenchyma to the vascular lumen, in which they signal to tethered, rolling lymphocytes? No definitive answers for these questions have been forthcoming. Chemokines produced by BBB endothelium during EAE include the lymphoid ‘homeostatic’ chemokines CCL19 and CCL21 [42,43]. In vitro, these chemokines trigger adhesion of encephalitogenic CCR7-positive T lymphocytes to inflamed brain vessels in Stamper-Woodruff assays (see supplementary Table S1 online). Their impact in T-lymphocyte recruitment across the BBB in vivo during EAE remains to be investigated. Preliminary studies of MS tissues have

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not identified CCL19 or CCL21 immunoreactivity in lesional vasculature [44]. Numerous other chemokines are produced in the inflamed brain, both in EAE and in MS, and encephalitogenic lymphocytes express a substantial menu of chemokine receptors. Mice devoid of either CCR1 or CCR2 are relatively resistant to active-immunization EAE, largely as a result of impaired monocyte recruitment, and T lymphocytes from CCR2K/K animals can mediate adoptive transfer EAE. Antibody-mediated blockade of CXCL10, the ‘Usual Suspect’ for inflammatory lymphocyte recruitment, provides conflicting results and CXCL10-deficient mice exhibit reduced threshold of EAE susceptibility [45–47]. Dissecting chemokine translocation across endothelium is in its infancy and, for the BBB, is embryonic. Mechanisms proposed include: intercellular diffusion (which seems unlikely at the BBB); transport by non-signalling ‘interceptors’, such as the Duffy antigen receptor for chemokines (DARC); and transport by signalling receptors, such as CCR2, which are present in vitro on cerebrovascular endothelium [48]. However, CCL2 immunoreactivity is localized to the abluminal aspect of vessels in transgenic mice that overexpress this chemokine and develop monocyte infiltrates [49]. Furthermore, CCR2 immunoreactivity has not been associated with cerebrovascular endothelium in vivo. Complexities became evident on analyzing other transgenic mice that overexpress T lymphocyte-specific chemokines, such as CCL19, CCL21 and CXCL10, in the CNS [50–52]. The phenotypes of these models are surprising: none accumulate lymphocytes in the brain; some develop neutrophilic inflammation; others are immunohistologically indistinguishable from wild-type mice and none exhibit neurobehavioral abnormalities. Answering problematic questions that surround the roles of chemokines in lymphocyte accumulation in the inflamed and healthy CNS might elucidate immunotherapeutic strategies. Firm adhesion and diapedesis In several studies, the adhesion molecules ICAM-1 and VCAM-1 were upregulated on CNS microvascular endothelial cells during EAE [12,53,54]. The expression of VCAM by human cerebral vasculature remains controversial, with one positive study [55] but two studies showing VCAM on activated microglia but not on endothelium [22,56]. With one exception [57], MAdCAM-1 (mucosal addressin cell adhesion molecule-1) expression has not been detected at the BBB during EAE [53]. Perivascular inflammatory cells surrounding ICAM-1- and VCAM-1positive murine venules stain positive for LFA-1 (aLb2-integrin) and a4b1-integrin, the ligands for ICAM-1 and VCAM-1, respectively, but not for L-selectin or a4b7-integrin. Antibodies to a4b1-integin inhibit or reverse EAE in different animal models [24,58,54,59]. Frozen section adhesion assays on EAE brains demonstrate the binding of lymphocytes, through LFA-1 and the a4-integrins, a4b1 and a4b7, to their respective endothelial ligands ICAM-1 and VCAM-1, expressed on the inflamed cerebral vessels within the tissues [53,58]. Furthermore, adhesion of encephalitogenic T lymphocytes to brain endothelial cells www.sciencedirect.com

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in vitro is mediated by a4–VCAM-1 and LFA-1–ICAM-1 interactions [60]. Finally, surface expression of a4-integrin on neuroantigen-specific T-cell clones correlates with their encephalitogenicity [54]. Taken together, these observations clearly implicate a4b1–VCAM-1 and probably also LFA-1–ICAM-1 in CNS inflammation during EAE. The involvement of a4b7–MAdCAM-1 in EAE remains controversial because neutralizing a4b7 antibodies do not inhibit EAE in SJL mice [24]. By contrast, b7-integrindeficient mice exhibit mild EAE [61], however, it was not addressed whether or not a4-integrin expression levels on leukocytes are generally lower in b7-integrin-deficient mice, which would explain these varying findings. MAdCAM-1 antibodies inhibit EAE in C57Bl/6 mice [62], which is unexpected because MAdCAM-1 is not detected on CNS endothelium in C57Bl/6 mice. Thus, the precise role of MAdCAM-1 and a4b7-integrin in EAE pathogenesis remains to be clarified. Finally, although the connecting segment 1 (CS1)-containing fibronectin isoform binds a4-integrins, its potential involvement in EAE or MS has been virtually ignored [63,64]. The contribution of the LFA-1–ICAM-1 interaction in inflammatory cell recruitment across the BBB during EAE remains uncertain. Antibody-inhibition studies in EAE produced contradictory results, ranging from inhibiting EAE to increasing the severity of EAE [65–68]. These confusing results might stem from the ability of some reagents to alter LFA-1–ICAM-1 interactions within immunological synapses and thus lymphocyte activation (reviewed in Ref. [69]). By contrast, blocking antibodies against LFA-1 and ICAM-1 consistently impair T-lymphocyte–cerebrovascular endothelial interactions in vitro [70,71], and studies using ICAM-1 or ICAM-1- and ICAM-2-deficient endothelium showed that ICAM-1, and to a lesser degree ICAM-2, is essential for T-lymphocyte migration across brain endothelium in vitro [72,73]. Evaluation of this axis in MS shows consistently that ICAM-1 (but not cytokine-unresponsive ICAM-2) is upregulated dramatically on inflamed vessels in MS lesions [74,75] and that essentially all infiltrating haematogenous leukocytes in MS lesions are LFA-1C [75].

Does the BBB prompt transcellular leukocyte migration? During diapedesis across the inflamed BBB, leukocytes migrate by a transcellular pathway through endothelial cells, leaving tight junctions intact (reviewed in Ref. [76]). Transcellular migration of leukocytes might be considered a specialization of the nervous system endothelial cells that are connected by complex tight junctions. Recent in vitro studies confirmed transendothelial leukocyte migration involving LFA-1 and ICAM-1 [77]. It remains pertinent to address the involvement of junctional molecules, such as junctional adhesion molecule-A (JAM-A) and platelet–endothelial cell adhesion molecule-1 (PECAM-1), in CNS inflammation. PECAM-1deficient mice with EAE have increased CNS infiltrates [78], accompanied by exaggerated, prolonged cerebrovascular permeability, and PECAM-1-deficient vascular elements in vitro recover junctional integrity slowly following inflammatory challenge [78], linking the

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regulation of vascular permeability to leukocyte transmigration. JAMs are localized in endothelial tight junctions (reviewed in Ref. [79]). Blocking JAM-A inhibits leukocyte diapedesis in vitro and leukocyte entry into the meninges [80]. In a second study, blocking JAM-A in vivo with the same reagents failed to prevent leukocyte influx during meningitis [81]. Therefore, the involvement of JAM-A in leukocyte migration across meningeal vessels remains undefined. Recently, CD99, a unique highly O-glycosylated protein expressed on leukocytes, was localized in endothelial cellto-cell-contacts [82]. CD99 antibodies inhibit the migration of encephalitogenic T lymphocytes across brain endothelial monolayers in vitro, suggesting the involvement of CD99 in T-lymphocyte recruitment across the BBB in vivo [83]. The choroid plexus during CNS inflammation ICAM-1 and VCAM-1 were detected previously on inflamed choroid plexus epithelium (CPE) [84,85]. These molecules mediate inflammatory cell binding to CPE in Stamper-Woodruff assays [21]. Both IHC and in situ hybridization demonstrated upregulation of VCAM-1 and ICAM-1 and de novo expression of MAdCAM-1 in the choroid plexus during EAE. Ultrastructural studies localized ICAM-1, VCAM-1 and MAdCAM-1 to the apical surface of CPE cells and their absence on the fenestrated endothelial cells within the choroid plexus parenchyma [86]. Interestingly, DCs are recruited across the CPE during EAE [87], further supporting the immune function of this organ [88]. Of great potential significance, lymphoid-like aggregates might form within the meninges during sustained neuroinflammation, as observed in chronic EAE or progressive MS [89,90]. In principle, these structures, whose formation is guided more by the homeostatic than by the inflammatory chemokines or chemokine receptors, could be relatively resistant to standard approaches to immunomodulation and their molecular constituents could provide highly attractive targets for the treatment of the most intractable aspects of MS. New developments EAE studies summarized earlier conclude that a4–integrin has a central role in leukocyte migration across the blood– tissue barriers of the CNS. Clinical MS trials confirm this hypothesis: natalizumab (humanized anti-a4 integrin antibodies) has produced the most impressive reduction of MS inflammatory disease activity (relapses; inflammatory gadolinium-enhancing lesions or new T2-bright magnetic resonance imaging lesions) yet reported [91] and was rapidly approved for use in patients. Post-release, three of approximately three thousand patients that received natalizumab during clinical trials for treating MS, Crohn’s disease and rheumatoid arthritis developed progressive multifocal leukoencephalopathy (PML), a fatal or catastrophic re-activation of JC virus within CNS glial cells. Because the population incidence of PML is 3 per 106 per year and virtually every previous PML case occurred in individuals with evident, generalized immunosuppression, PML was clearly treatment-related www.sciencedirect.com

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in the natalizumab-treated cohort, which, otherwise, exhibited a comprehensive lack of opportunistic infections. These shocking events led to the voluntary suspension of use of the agent as a treatment for MS and halted ongoing natalizumab trials. It remains to be determined whether PML in these patients resulted from release of bone marrow cells harbouring pathogenic virus, alone or in combination with impaired CNS immunosurveillance. Several lessons can be provisionally drawn from this bewildering and disappointing occurrence: (i) leukocyte trafficking, and a4 integrins in particular, are validated treatment targets for MS; (ii) pathogenic mechanisms for PML in natalizumab-treated patients need to be clarified urgently; (iii) continued research to understand precisely the involvement of adhesion and signalling molecules involved in the recruitment of leukocytes across the highly specialized blood–CNS barriers is crucial, as we seek to capitalize on unrealized opportunities and avoid unexpected pitfalls. Conclusions, implications and complications Immune cells patrol the CNS to perform immunosurveillance. However, for this purpose they migrate in various ways to different destinations. Lymphoblasts cross the endothelial inflamed BBB to ‘meet’ APCs, such as perivascular DCs of the CNS parenchyma [31]. Without antigen-triggered activation these cells will not persist, nor traverse the glia limitans into the CNS parenchyma. Such invasion only occurs under pathological conditions, during which ‘traffic signals’ are altered on the BBB endothelium and astroglial chemokines promote leukocyte penetration into the CNS parenchyma. A different process occurs in the subarachnoid space, in which dendritic elements, such as the Kolmer’s (epiplexus) cells, reside on the luminal surface of the choroid plexus, and present antigen to CSF lymphocytes that are carrying out immunosurveillance. During MS attacks, CSF leukocyte counts are elevated reliably, indicating that the choroid plexus and meninges support increased inflammatory trafficking. Molecular mechanisms involved in inflammatory cell entry clearly differ, depending on the stimulus and CNS site. Present observations suggest that there might be differential leukocyte–endothelial interactions in the CNS white matter versus grey matter, and spinal cord compared with brain, as well as meningeal vessels versus parenchymal vessels. Update The New England Journal of Medicine recently carried case reports of all three confirmed PML patients from natalizumab clinical trials [95–97]. These reports were supplemented with two editorials, one humbly acknowledging the courage of patients that enter clinical trials [98]; the other editorial [99] proposed an hypothesis: that PML occurs in natalizumab recipients because of deficient immunosurveillance at sites of latent infection and, possibly, the CNS as well. However, the case reports, with their abundant detail, illustrate a complex spectrum of poorly understood virological and host factors in these patients, which render the defective immunosurveillance

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hypothesis premature. One MS patient without unusual features developed PML and succumbed rapidly to her disease [95]. The second MS patient, also without unusual features, was noted to have atypical magnetic resonance imaging (MRI) changes for MS and was taken off natalizumab. Within months, he developed inflammatory MRI changes in regions of PML involvement and neurological deterioration, possibly indicating immune reconstitution syndrome. Administration of cytosine arabinoside (which does not cross an intact BBB) was associated with sustained and progressive improvement. The third case, with Crohn’s disease [97], was attended by physicians sufficiently astute to have banked serum for five years preceding his fatal PML. It was therefore possible to correlate precisely the development of JC viremia with administration of natalizumab. These well documented cases will assist with elucidating the pathogenesis of PML in natalizumab recipients and help define whether deficient immunosurveillance, or alternative mechanisms, are responsible. Acknowledgements The B.E. laboratory has been supported by Astra Zeneca, the German Research Foundation (SFB 293, SFB 629), the German Ministry for Education and Research, the Swiss National Science Foundation (3100A0–104096), GlaxoSmithKline and the Swiss Multiple Sclerosis Society. The R.M.R. laboratory has been supported by the US NIH (NS32151; NS38667; NS36674; TW6012), the Charles A. Dana Foundation, the Nancy Davis Center Without Walls and with fellowship and pilot project awards from the National Multiple Sclerosis Society. We regret that much important work could only be cited incompletely or not at all due to space limitations.

Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.it.2005.07.004

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