Factors modifying the migration of lymphocytes across the blood–brain barrier

International Immunopharmacology 1 Ž2001. 2043–2062 www.elsevier.comrlocaterintimp

Review

Factors modifying the migration of lymphocytes across the blood–brain barrier K. Alun Brown Department of Immunobiology, 3rd Floor New Guy’s House, Guy’s Hospital, London SE1 9RT, UK Received 26 February 2001; received in revised form 19 June 2001; accepted 19 June 2001

Abstract Characterising the factors that control the entry of leucocytes into tissue in response to inflammatory or microbial insult continues to generate considerable interest. Of all the tissues studied it is probably that of the CNS which is the most fascinating because of the specialised properties of its blood vessel walls, which constitute the blood-brain barrier ŽBBB.. In health, very few leucocytes penetrate the BBB but in disorders such as MS the barrier becomes compromised with the result that there is an intense infiltration of the CNS by T lymphocytes whose subsequent activity appears to underlie the onset and progression of disease. The purpose of this article is to summarise and assess recent literature pertaining to how lymphocytes bind to cerebral endothelial cells, migrate across the blood vessel walls and enter the CNS parenchyma. Particular emphasis is devoted to the cellular and molecular aspects of these events and addressing the questions of whether certain subsets of circulating T lymphocytes are more favourably disposed than others to CNS infiltration and whether entry is dependent upon the initial expression of distinct groups of adhesion molecules and upon the generation of chemotactic factors. This article also focuses upon identifying the key stages of lymphocyte migration across the BBB and their susceptibility to antagonism by therapeutic agents. It is intended that the review will provide a useful source of information and offer additional insights into the mechanisms controlling lymphocyte passage across the BBB during pathological disturbance. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Lymphocyte; Blood; Brain

1. Introduction Since the early 1980s, there has been an unprecedented increase in the volume of literature pertaining to the factors controlling the migration of leucocytes across blood vessel walls in response to infections or inflammatory insult w1,2x. The plethora of information has provided additional considerations into the mechanisms by which leucocytes penetrate the walls of blood vessels in the central nervous system ŽCNS. whose resistance to leucocyte extravasation and plasma permeability is aptly termed the blood–brain

barrier ŽBBB.. The unique barrier features of microvessels within the brain and spinal cord used to be ascribed to the specialised properties of endothelial cells and to the formation of tight junctions between the cells with a high electrical resistance. It is now recognised that the barrier function of CNS vessels arises from an integrated dynamic structure that is comprised of endothelial cells, pericytes, astrocytes and macrophages. For many years, the brain was regarded as immunologically privileged because isolation from the immune system by the BBB led to an improved

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survival of allografts in this organ in comparison with more conventional sites w3x. Complementary to this view was the sparse number of antigen-presenting cells in brain tissue, an extremely low expression of MHC Class II molecules and the apparent absence of a conspicuous lymphatic system that would allow antigen to be delivered to secondary lymphoid tissue and enable infiltrating lymphocytes to return to the circulation. However, with the revelation that cervical lymphatics provide communication between the brain and lymphoid tissue w4x and that small numbers of lymphocytes traverse the BBB, it is now accepted that the brain is subjected to limited immunological surveillance w5x. Irrespective of antigen specificity, activated lymphocytes readily transverse blood vessels in normal brain and spinal cord as demonstrated by their appearance in the CNS within 24 h of injection into a naıve ¨ animal recipient w6,7x. Should the relevant CNS antigen be encountered, then the activated cells will make the appropriate response and reside in the tissue, but in the absence of antigen, they will leave the CNS via the lymphocytes before returning to the circulation. Sites of microbial entry into the body, namely the lung, gut, urinary tract and skin, frequently accommodate large-scale invasion by pathogen seeking leucocytes. Most infections of the CNS emanate from the circulation and it is for this reason, more than any other, that is responsible for the evolution of the unique barrier properties of cerebral vessel walls. Occasionally, the BBB succumbs to microbial entry with severe pathological consequences such as measles infection in sub-acute sclerosing panencephalomyelitis and the meningoencephalitis associated with HIV infection. Elimination of pathogens by leucocytes is frequently accompanied by limited tissue damage due to over-exuberance of the cells in their extracellular release of lytic factors. Whereas most non-CNS tissue is capable of sustaining a small degree of damage that does not compromise its function, such an event in the CNS may prove to be catastrophic because the integrated network of neurons has a low regenerative capacity with little connective tissue support. In multiple sclerosis ŽMS., large numbers of leucocytes infiltrate the CNS in the absence of any apparent infection to produce widespread tissue insult. Since most of the information concerning the migration of lymphocytes across

the BBB has emerged from the study of MS and its animal model, experimental allergic encephalomyelitis ŽEAE., this review will focus upon T lymphocyte extravasation in relation to demyelination.

2. Pathogenetic features of multiple sclerosis Multiple sclerosis is regarded as a T lymphocytedependent chronic inflammatory disease of the CNS characterised by demyelinated plaques with glial scar formation w8–10x. The contribution of B lymphocytes to the disease pathophysiology is adequately summarised elsewhere w11x. Destruction of myelin leads to a diminished nervous conduction in the brain and spinal cord, subsequent motor and sensory disturbances and disability. The disease normally commences with a relapsing–remitting phase, later followed by a more progressive course. Both an infectious w12x and an autoimmune aetiology are suspected w13x with the migration of reactive T lymphocytes across the BBB being central to the process of demyelination. The plaques are primarily located in the periventricular white matter and the cervical area of the spinal cord. Recognition of myelin antigens by cytotoxic CD8q T cells may lead to direct damage of the myelin or to the oligodendrocytes which produce the myelin, whereas CD4q T cell recognition could mediate indirect destruction by the release of cytokines which recruit and activate either resident microglia, astrocytes or infiltrating monocytes w14,15x. Activation of myelin-reactive T lymphocytes could be a consequence of molecular mimicry. The considerable homology that exists between the major constituent of myelin, major basic protein ŽMBP. and several common viruses such as measles, Epstein–Barr virus and adenovirus is thought to be sufficient for the microbes to elicit stimulation of MBP-specific T cell clones from MS patients w16x. Hence, it is proposed that disease relapses in MS, which often occur after viral infection of the respiratory or gastrointestinal tract, are precipitated by viral-induced immunological cross-reactivity with myelin. A distinct feature of MS brain is the appearance of cuffs of lymphocytes around small blood vessels. Infiltrating CD4q T lymphocytes tend to predominate in early CNS lesions and CD8q T lymphocytes

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at later stages w15x. Within active demyelinating lesions, the vast majority of cells resemble macrophages. Of the infiltrating CD4q T lymphocytes most express the phenotype of Th1 lymphocytes in that they produce the pro-inflammatory cytokines interferon-g ŽIFNg . and interleukin-2 ŽIL-2. in contrast to Th2 cells which secrete the B lymphocyte differentiation cytokines IL-4 and IL-5 and the anti-inflammatory IL-10. Compelling evidence for a pathogenic role of Th1 cells in MS emanated from a clinical trial of IFNg , which had to be terminated because of the clinical deterioration of the participating patients w17x. Although the distribution of circulating effector CD4q and effector CD8q T lymphocytes is not altered during the clinically active stage of MS w18x, there is an increase in the numbers of CD4q T lymphocytes and CD8q T lymphocytes expressing IFNg mRNA w19x. Other possible participants of demyelination are gd T lymphocytes, which recognise heat shock proteins associated with CNS injury w20x. In MS, there

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are increased frequencies of gd T lymphocytes in the blood and CSF, which accumulate in MS plaques and in culture lyse oligodendrocytes w21–23x. Most of the cells express the Vg 2 T cell receptor; the remainder, the Vg 1 T cell receptor. The latter subgroup predominates in the CSF of MS patients w23x with Vg 2 T cells being more confined to lesions w24x. In MS patients, it is the Vg 2 subset that is activated and which produces large amounts of proinflammatory cytokines and chemokines w25x. Current speculation is that gd T lymphocytes contribute to the chronicity of lesions. Studying the pathological features of MS and EAE which is a CD4q Th1-mediated autoimmune disease w26x has provided valuable information concerning the mechanisms that control the passage of lymphocytes across the BBB. In rodents, EAE is induced by active immunisation with myelin-derived peptides or by the passive transfer of T lymphocytes specific for myelin peptides. Demyelination arises from the recognition of target antigen by infiltrating

Fig. 1. Factors considered to promote the migration of lymphocytes across the blood–brain barrier in multiple sclerosis.

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autoreactive CD4q T lymphocytes and the release of Th1 cytokines. Interestingly, antigen-specific T lymphocytes account for less than 4% of the total leucocyte infiltrates w27x with the extent of CNS inflammation being related to the degree of leucocyte infiltrates across the BBB w28x. 3. Migration of lymphocytes across the blood– brain barrier It is likely that factors contributing to circumvention of the normal restrictive properties of the BBB by lymphocytes are operating at several stages as outlined in Fig. 1. Certain subsets of lymphocytes may already be ‘primed’ to bind to cerebral vessel walls adjacent to areas of tissue insult by expressing on their surface, adhesion-promoting determinants that recognise endothelial ligands whose own expression is upregulated by inflammatory factors generated within the CNS. Binding could also be facilitated by the activity of soluble factors in the circulation. Upon contact with endothelial cells, the lymphocytes may become responsive to chemotactic concentration gradients that direct the cells through the remaining constituents of the vessel walls into the perivascular ŽVirchow–Robin. space where some accumulate and others move into the white matter parenchyma. Let us now examine the evidence supporting each of these proposals. 4. Adhesion molecules Lymphocyte binding to blood vessel walls depends upon the sequential interaction of adhesion molecules on the leucocyte surface with corresponding ligands expressed on endothelial cells. A comprehensive description of the structure and function of adhesion molecules is provided by several recent articles w1,2,29x. Before considering the relative merits of adhesion molecules to the attachment of lymphocytes to constituents of the BBB, this review will briefly outline the current understanding of the main forms of adhesion molecules. There are three principal groups: the integrins, members of the immunoglobulin gene superfamily and the selectins. Integrins are heterodimers with an a-subunit non-covalently linked to a b chain. There are almost

20 heterodimers derived from the association of approximately 16 a chains and eight b chains. Based upon the b chain usage, integrins are classified into several subfamilies, which are generally confined to leucocytes. Some forms of a chain may associate with a particular form of b chain, whereas several may link to more than one type of b chain. The term integrin was coined to define molecules on the cell surface that linked the intracellular cytoskeleton with constituents of the extracellular matrix. Levels of integrin expression vary with stages of cellular differentiation and activation. Divalent cations are essential for integrin binding. The b 1 integrins are also referred to as the very late antigen ŽVLA. family since they were found to appear late on the surface of lymphocytes following activation, though it is now known that VLA-4 and VLA-6 are expressed on resting lymphocytes. As shown in Table 1, each b 1 integrin recognises one or more of the proteins of the extracellular matrix with the excep-

Table 1 Lymphocyte adhesion molecules implicated in binding to blood vessel walls Adhesion molecule

Other designations

b 1 integrins a1b 1 VLA-1 CD49arCD29 a 2b1 VLA-2 CD49brCD29 a 3b 1 VLA-3 CD49crCD29 a 4b 1 a 5b 1 a 6b 1

VLA-4 CD49drCD29 VLA-5 CD49erCD29 VLA-6 CD49frCD29

b 2 integrins aLb2 LFA-1 CD11arCD18 a Mb2 Mac-1 CR3 CD11brCD18 aXb2 p150,95 CR4 CD11crCD18 b 7 integrins a 4b 7 CD31 CD44

PECAM-1

CD62L

L-selectin

Ligands

collagen, laminin collagen, laminin collagen, fibronectin, laminin fibronectin, VCAM-1 fibronectin laminin

ICAM-1, ICAM-2 ICAM-1, C3Bi fibrinogen? C3Bi

fibronectin, VCAM-1, MadCAM-1 CD31 a vb 3 fibronectin, collagen, hyaluronic acid, glycosaminoglycan GlyCAM-1, MadCAM-1, CD34

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tion of VLA-4, which also binds to the vascular cell adhesion molecule-1 ŽVCAM-1., otherwise known as CD106 Žsee below.. The b 2 integrins are referred to as the leucocyte integrins with CD11arCD18 expressed on all classes of leucocytes and CD11br CD18 and CD11crCD18 generally confined to phagocytic cells Žneutrophils and monocytes. and some subsets of lymphocytes. Integrins are important for promoting the firm interaction of leucocytes with endothelial cells. Members of the immunoglobulin gene superfamily that are associated with endothelial cells include intercellular adhesion molecule-1 ŽICAM-1., also referred to as CD54, which has five Ig domains; ICAM-2, which has two domains and vascular cell adhesion molecule-1 ŽVCAM-1., otherwise known as CD106, which has six domains. Resting endothelial cells constitutively express far more ICAM-2 than ICAM-1. Upon stimulation with cytokines that include IL-1, TNFa and IFNg , there is a large increase in the surface expression of ICAM-1 due to de novo synthesis. ICAM-2 is refractory to upregulation by cytokines. The first Ig domain of ICAM-1 and ICAM-2 interacts with CD11a, the third domain of ICAM-1 with CD11b. Hence, both ICAM-1 and ICAM-2 augment the attachment of all leucocytes with ICAM-1 having the more prominent role at sites of inflammation. VCAM-1 is normally absent from the surface of unstimulated endothelial cells but its expression is also upregulated upon exposure to TNFa , IL-1, IL-4, but not IFNg w30x. Through its recognition by VLA-4, the induced VCAM-1 supports the attachment of lymphocytes and monocytes. Platelet–endothelial cell adhesion molecule-1 ŽPECAM-1. or CD31 is another member of the immunoglobulin superfamily that is expressed on endothelial cells and also platelets and leucocytes w31x. PECAM-1 is mainly confined to endothelial cell junctions where it promotes leucocyte attraction and transendothelial migration by homophilic adhesive interactions. A synopsis of the contribution of integrins to immune-mediated disease of the CNS is provided by Archelos et al. w32x. Selectins participate in the early tethering and rolling of leucocytes on the endothelial surface. They are single transmembrane polypeptides, which possess an N-terminal lectin domain that recognises distinct carbohydrate moieties and a cytoplasmic tail,

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which evokes signal transduction. With the exception of memory lymphocytes, L-selectin is present on nearly all leucocytes. L-selectin participates in the tethering of leucocytes to blood vessel walls and in the ‘homing’ of lymphocytes to secondary lymphoid tissue. Upon cellular activation and during extravasation, L-selectin is shed from the surface: the functional significance of this event is not known. Both P- and E-selectin appear on the surface of activated endothelial cells. Within minutes of endothelial stimulation by inflammatory factors such as histamine and thrombin, P-selectin is rapidly translocated to the cell surface from vesicles known as Weibel Palade bodies. Induction of E-selectin expression is dependent upon de novo synthesis instigated by pro-inflammatory cytokines. The following is a general description of the adhesive stages involved in the migration of blood leucocytes to sites of inflammatory insult. The initial contact of leucocytes with endothelial cells is mediated by L-selectin on leucocytes and E- and P-selectin on endothelial cells recognising distinct carbohydrate moieties on opposing cells. Selectins serve to tether leucocytes to endothelial cells so as to allow rolling in the direction of blood flow. In the absence of other adhesion molecules, selectin-mediated adhesion becomes transient with the loosely adherent cells being returned into the circulation. Integrins control the firm adhesion of leucocytes to endothelium. When leucocytes roll along endothelium, their integrins become activated following cell contact with inflammatory stimuli that include plateletactivating factor ŽPAF., the complement peptide C5a and chemokines such as interleukin-8 ŽIL-8. w33x expressed on the endothelial surface. This activation may involve an increased expression andror conformational changes in the integrins resulting in increased affinity for the endothelial ligands, whose own expression is upregulated by pro-inflammatory cytokines. These strong molecular interactions result in the spreading of leucocytes on the endothelial surface. For lymphocytes, this latter event is dependent upon the recognition of ICAM-1 by the b 2 integrin CD11arCD18 and of VCAM-1 by the b 1 integrin VLA-4. CD44, is a proteoglycan whose polypeptide chain exists in various isoforms due to alternative exon splicing w34x. Its principal ligand is glycosaminoglycan hyaluronate, which is a con-

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stituent of the extracellular matrix and contributes to the rolling of activated lymphocytes on endothelium w35x. CD44 is believed to participate in the extravasation of lymphocytes into inflammatory lesions w36x, homing to high endothelial venules Žsee below. w37x and the presentation of chemokines to leucocytes w38x. Upon firm contact with endothelial cells, the lymphocytes will subsequently migrate through the endothelium and the remainder of the blood vessel wall by a series of additional adhesive interactions that include recognition of the proteins of the extracellular matrix by the b 1 integrins. The directed migration of lymphocytes to the focus of the inflammatory lesion is in response to the activity of chemotactic factors such as C5a and chemokines. In their quest for foreign antigens, lymphocytes are constantly moving between blood and tissue. The generally held view was that naıve ¨ lymphocytes, i.e. lymphocytes that have not encountered antigen, circulate between blood and secondary lymphoid tissue, whereas the recirculation of memory lymphocytes is confined to non-lymphoid tissue. However, recent studies reveal that both naıve ¨ and memory T lymphocytes readily migrate into lymphoid and nonlymphoid tissue w39,40x. Secondary lymphoid tissue such as spleen, lymph nodes and the less organised mucosal-associated lymphoid tissue contain vast numbers of naıve ¨ lymphocytes. They provide an environment where foreign antigens are sequestered, presented to the appropriate naıve ¨ lymphocytes, which upon recognition clonally proliferate into memory cells. Upon future contact with antigen, the memory T cells will exert their effector activities Že.g. killing of virus-infected cells, release of cytokines to activate macrophages.. Therefore, it is appropriate that memory T lymphocytes encounter the antigens at their source of entry into the body, e.g. skin, respiratory and GI tract, or at sites where pathogens eventually reside. Circulating naıve ¨ lymphocytes enter lymphoid tissue by their ‘homing’ receptors recognising specific ligands, termed addressins, on the surface of post-capillary venules w2x. Within secondary lymphoid tissue but not spleen, the venules that support lymphocyte migration are referred to as high endothelial venules ŽHEV. because their endothelial cells acquire a characteristic large, plump, cuboidal morphology. L-selectin is a homing receptor for lymphocytes which recognises the pe-

ripheral lymph node addressin ŽPNAd. on HEV and the addressin glycosylation-dependent cell adhesion molecule-1 ŽGlyCAM-1. which is synthesised by HEV of lymph nodes. So far, it is only in the mouse that L-selectin also binds to the mucosal addressin cell adhesion molecule-1 ŽMadCAM-1., which is expressed on the HEV of Peyer’s patches and mesenteric lymph nodes.

5. Expression of vascular adhesion molecules at the BBB during demyelination Elevated levels of ICAM-1 are a feature of the microvasculature adjacent to demyelinating lesions in EAE w41,42x and in MS lesions w43–45x. During the clinical course of EAE, the expression of ICAM-1 on cerebral endothelial cells is low prior to lesion development, increased in active lesions and decreased in remission w46x. ICAM-1 expression is upregulated by pro-inflammatory cytokines that are present in MS lesions w47,48x. Of particular note is TNFa w49x, which increases the adhesiveness of endothelial cells for lymphocytes w29x and promotes lymphocyte migration across the BBB w50,51x. In EAE, the intrathecal injection of TNFa initiates perivascular cuffing and demyelination w52,53x, whereas anti-TNFa antibodies abrogate disease progression w54,55x. Not only is TNFa efficient at increasing ICAM-1 expression on endothelial cells, but it is also effective at inducing the endothelial expression of VCAM-1 that appears on blood vessels within MS plaques w42x and which is recognised by T lymphocytes bearing the b 1 integrin, VLA-4 w3x. It therefore appears that cerebral endothelial cells behave similarly to peripheral endothelial cells in their upregulation of ICAM-1 and VCAM-1 by inflammatory cytokines and subsequent support of lymphocyte binding. However, such a view is not universally accepted as illustrated by the failure of one study to identify VCAM-1 on endothelial cells in MS lesions w56x. Based upon the successful demonstration that anti-ICAM-1 and anti-LFA-1 antibodies improved cardiac allograft survival by preventing lymphocyte infiltration w57x, a similar experimental approach was applied to EAE. Initial claims that anti-ICAM-1 antibodies suppressed disease progression w58x were tempered by other reports of disease exacerbation

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following administration of the antibodies w59,60,61x. Such an effect may be a peculiarity of the EAE model since anti-ICAM-1 antibodies did not modify the neuropathology of demyelinating disease in Theiler’s murine encephalomyelitis virus-infected mice w61x. On the basis that there is neither an abnormal expression of E- and P-selectin nor an increased distribution of lymphocytes with receptors for these ligands in MS tissue, it seems that these selectins are not contributing to the passage of lymphocytes across the BBB w62x. Both E- and P-selectin play a pivotal role in the initial tethering of neutrophils to endothelium and therefore, their low expression on endothelial cells in close proximity to MS lesions may explain the relative exclusion of neutrophils from infiltrating leucocytes. As outlined earlier, lymphocytes home to lymph nodes and mucosal-associated lymphoid tissue by recognising addressins such as MadCAM-1 and GlyCAM-1 on HEVs. The specific homing of blood lymphocytes to the CNS in MS is an intriguing concept but one which lacks strong experimental foundation. High endothelial cells have not been consistently demonstrated in the CNS vessels of EAE or MS, and it remains contentious as to whether such vessels are particularly associated with the expression of addressins w63,64x. In the absence of any antibody blocking studies, it is unlikely that recognition of the known vascular addressins is a necessary prerequisite for lymphocyte entry into the CNS. Furthermore, the overall impression of the above reports is that there is no convincing evidence to advance the proposal that passage of lymphocytes across the BBB is initially controlled by the selective singular or combined expression of known vascular adhesion molecules.

6. Phenotypic characterisation of blood lymphocytes that enter the CNS The purported preferential homing of naıve ¨ T lymphocytes to lymphoid tissue and the selective extravasation of memory T lymphocytes may be reflected in their differential expression of adhesion molecules. In general, the expression of b 1 and b 2 integrins is greater on memory T lymphocytes than naıve ¨ lymphocytes w65,66x, whereas naıve ¨ T cells

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possess a higher expression of L-selectin. Most of the mononuclear cells that infiltrate CNS lesions are memory T lymphocytes w67,68x which is commensurate with the high binding affinity of these cells for cytokine-activated endothelium. Let us now examine the evidence that suggests that in MS, certain blood lymphocytes are predisposed for CNS extravasation. As outlined earlier, immunological surveillance of normal brain is a prerogative of activated lymphocytes w69,70x, which have an increased representation in MS blood. Work from our laboratory shows that blood T lymphocytes from patients with MS, particularly those with evidence of a recent clinical relapse, adhere in greater numbers to endothelial cells stimulated with TNFa than lymphocytes from healthy subjects and patients with other inflammatory disorders w71,72x. This augmented adhesion is a characteristic of CD4q T lymphocytes which are more efficient than CD8q T cells in binding to blood vessel walls exposed in tissue sections of normal human brain w73x. Moreover, our recent studies reveal MS lymphocytes to exhibit a supranormal binding to blood vessel walls in active lesions of MS brain. Compatible with these findings is the augmented binding of blood mononuclear cells from patients with MS to blood vessel walls in normal brain tissue w74x An adhesion molecule credited with a prominent role in promoting the extravasation of T lymphocytes across the BBB during inflammatory insult is VLA-4. Antibodies against VLA-4, but not other b 1 or b 2 integrins, prevented lymphocyte attachment to blood vessel walls in brain sections from animals with EAE. Following administration of the antibodies to animals 2 days after the induction of EAE, paralysis was arrested in the majority of animals and its severity reduced in the remainder w75x. Anti-VLA-4 antibodies were later shown to delay disease onset, decrease disease severity w76–78x and impair CD4q T cell entry into the CNS parenchyma. The observation that encephalitogenic clones of T cells with a high expression of VLA-4 readily traversed the BBB led to the claim that VLA-4 expression was critical to the migration of activated T lymphocytes into the CNS w79x. This view is not unanimously accepted w80x, and it has been recently proposed that VLA-4 contributes to the maintenance of T lymphocytes in EAE lesions w81x. The distribution of lymphocytes

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expressing VLA-4 is raised in the blood and cerebral spinal fluid of MS patients w71,82x, and there is also an increased prevalence of CD4q T lymphocytes bearing VLA-4 and CD11a in patients with schizophrenia who have evidence of a disturbed BBB w83x. In an MS phase I clinical trial of anti-VLA-4 antibodies, clinical benefit was apparent during the early stages but overall disease progression was not modified w84x. Another adhesion molecule which may be contributing to the passage of T lymphocytes across the BBB is CD44, which is upregulated on activated lymphocytes w85x and on MS blood lymphocytes w86x. CD44 is present on T lymphocytes that undergo transendothelial migration w87x, but its expression is transiently downregulated following passage across cerebral vessel walls w88x. Antagonising CD44 expression prevents the development and severity of the CNS inflammation in EAE by interfering with the migration across the BBB of both antigen-specific and secondary recruited T lymphocytes w88,89x. Evidence is not so readily forthcoming to support the credentials of other adhesion molecules in EAE. In one study, the administration of anti-CD11a or anti-CD18 antibody improved disease activity w90x, whereas another found that anti-CD11a antibodies actually exacerbated disease severity w91x. Neither the entry of T cells into the CNS nor the course of EAE is modified by antagonising the expression of a 4b 7 w80x and L-selectin w92x, respectively. The above studies assessed the expression and contribution of known adhesion molecules to T lymphocyte–BBB interaction. They do not exclude the possibility that impacting upon these interactions are, as yet, uncharacterised adhesion determinants, some of which may be CNS restricted. Such optimism is fuelled by the recent report of a novel antigen on lymphocytes and endothelial cells that participate in cell trafficking across the BBB in EAE w93x. Also, all of the information presented so far relates to T lymphocytes, which utilise ab T cell receptors. As outlined earlier, the Vg 2 subset of gd T cells is also present at MS lesions. Activation of Vg 2 cells by IL-12 increases the surface expression of natural killer receptors ŽNKR., particularly the NKRP-1A form whose distribution on Vg 2 cells in MS blood is increased. Anti-NKRP-1 antibodies block the enhanced migration of Vg 2 but not Vg 1 cells across

endothelial monolayers w25x. As yet, there is no description of the adhesion molecule phenotype of Vg 2 T cells.

7. The contribution of soluble circulating factors to lymphocyte–blood vessel wall interaction Vascular adhesion molecules are upregulated on cytokine-stimulated endothelial cells whose continued activation or damage could lead to the release of soluble forms of the molecules w94x. In MS, raised levels of circulating ICAM-1 and VCAM-1 are believed to reflect disease exacerbation and BBB dysfunction w95x but such claims are refuted by studies which show neither elevated concentrations of the molecules in MS blood w96,97x nor any relationship to stages of disease activity w98x. This discordance may be a reflection of the clinical status of the patients. For example, increased levels of soluble ICAM-1 and VCAM-1 were noted in MS patients with primary progressive disease compared with patients with the relapsing–remitting or secondary progressive forms of the disease: no associations were apparent between their concentrations and the extent of disease disability and disease duration w99x. Evaluating the significance of soluble adhesion molecules to the clinical course and pathology of MS is complicated not only by conflicting reports of their levels in serum but also by interpreting the functional significance thereafter of their interaction with circulating T lymphocytes. Binding of the soluble molecules to their corresponding receptors on lymphocytes would be anticipated to competitively inhibit receptor recognition of their natural ligands on endothelial cells w100x. Indeed, cultures of human brain endothelial cells stimulated with TNFa released soluble forms of VCAM-1 that bound to mononuclear leucocytes and blocked their attachment to the endothelial monolayers w101x. However, lymphocyte ligation of soluble adhesion molecules could lead to an upregulation of other adhesion molecules as demonstrated by the increased expression of VLA-4 following binding of ICAM-1 to CD11a w102x and by the binding of anti-CD29 antibodies to T lymphocytes w103x. Blood levels of L-selectin are raised in MS patients with active disease w104x though whether soluble L-selectin modifies lymphocyte–endothelial

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interaction or merely provides evidence of leucocyte activation is not known. Other circulating factors that could be contributing to lymphocyte–endothelial cell interaction in MS are soluble forms of CD4 and CD8 w105x, PECAM-1 and cytokines, in particular, TNFa and its receptors w106x. High concentrations of soluble PECAM-1 occur in the sera of MS patients with active disease w107x, whereas increased levels of circulating TNFa are believed to be indicative of both the degree of disease activity w108x and of damage to the BBB w109x. Attributing TNFa a major pathogenic role in MS is tempered by the recent proposal that this cytokine acts as an anti-inflammatory factor in autoimmune-mediated demyelination w110x and that circulating levels in MS are within normal limits w111x. Current work in our laboratory shows that sera from patients with MS increases the binding of lymphocytes from normal healthy subjects to blood vessel wall in tissue sections of human brain. The effect is associated with the extent of the patient’s disease disability but not with the degree of disease activity, or with serum levels of TNFa , ICAM-1, VCAM-1, E-selectin and P-selectin. Adherence enhancing activity was further identified in sera from patients with other neurological disorders whose pathological basis implicates an abnormal interaction of lymphocytes with blood vessel walls.

8. Antigen presentation and the blood–brain barrier Once tissue damage has been initiated an array of inflammatory factors are produced to entice the extravasation of blood leucocytes. With regard to lymphocyte infiltration of the CNS, the question arises of whether migration across the BBB is essential to the onset of tissue insult or whether it is a secondary event that seeks to sustain and perpetuate the inflammatory response. In response to exogenous infection or exposure of myelin peptides, the appropriate antigen-specific T lymphocytes would be among the first cells to circumvent the BBB because of subsequent proliferation in peripheral lymphoid tissue and high representation in the circulating pool of activated lymphocytes. Cerebral entry is gained by activated lymphocytes recognising endothelial ligands, upregu-

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lated by cytokines produced by localised antigendriven tissue damage or by the recognition of antigen on blood vessel walls. Upon antigen interaction, the sensitised T lymphocytes secrete cytokines that recruit non-specific circulating leucocytes and local cells Žmicroglia, astrocytes. into the evolving plaque. An attractive hypothesis is that in the early stage of the disease, the immune response is targeted against a single antigen, but as CNS damage continues ‘cryptic’, new myelin epitopes appear that stimulate other autoreactive T lymphocytes w16x. The new antigenic epitopes generated within the CNS parenchyma diffuse into the CSF before draining into cervical lymph nodes where they sensitise T cells that enter the circulation w112x. Activation of naıve ¨ and memory CD4q T cells depends upon recognition of antigenic peptides presented by MHC II molecules. Once the T cell receptor has engaged antigen, co-stimulatory ligands provided by the binding of B7-1 and B7-2 on the antigen-presenting cell to CD28 on the lymphocyte are required for the full activation of the T lymphocyte. Without this stimulation, there will be T cell anergy w113x. Interferon-g activation of astrocytes w114x, smooth muscle cells w115x and perivascular macrophages w116x leads to the acquisition of MHC Class II molecules and to the presentation of myelin peptides. The close proximity of these cells to blood vessel walls provides an appropriate location for the rapid presentation of antigen to infiltrating T lymphocytes. Astrocytes even insert a foot-like process into the endothelium lining but it is the endothelial cells themselves with their large cumulative surface that are ideally positioned for antigen presentation. Stimulated human umbilical vein endothelial cells by IFNg induces the expression of MHC Class II but not co-stimulatory molecules w117,118x. Under appropriate conditions, endothelial cells present antigen to T lymphocytes w119,120x. Cerebral endothelial cells are normally devoid of MHC Class II molecules and B7-1 and B7-2. Although all of these molecules are induced on the surface of cultures of human brain endothelial cells, the activated cells are unable to sustain allogeneic CD4q T cell proliferation unless exogenous IL-2 is added w121x. It therefore appears that engagement of cerebral endothelial cells in antigen presentation depends upon the participation of activated T lymphocytes in close proximity to

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EAE w122,123x and MS lesions w124,125x. Moreover, the absence of the B7-1 and B7-2 from the surface of CNS endothelial cells and astrocytes during EAE strongly suggests that these cells are not actively engaged in antigen presentation w126x. Compatible with this finding is the proposition that brain endothelial cells are poor presenters of antigen w120x and that in EAE this function is predominantly ascribed to perivascular macrophages which possess a high expression of MHC Class II and co-stimulatory molecules w126x. Contact of memory CD4q T lymphocytes with antigen may not necessarily lead to cell activation. Cognate recognition of antigen presented by endothelial cells enhances the transmigration of antigen-specific T cell lines w127x, raising the intriguing consideration that the primary function of endothelial presentation of antigen is the recruitment of specific subsets of lymphocytes across the BBB. We will wait in anticipation to hear if brain endothelial cells pulsed with myelin peptides preferentially support the transendothelial migration of autoreactive T lymphocytes.

9. Chemokines For many years, the directed migration of lymphocytes into tissue was ascribed to the activity of chemotactic factors Že.g. C5a. that were equally effective for other leucocytes. With the advent of the chemokines, several molecules were identified with chemotactic activity specific for mononuclear leucocytes. Currently, scores of cytokines are defined in rodents and man w128x, and for the benefit of brevity, this section will focus upon those chemokines for whom evidence is available to support a role in the CNS extravasation of T lymphocytes. Chemokines possess a common structural motif which consists of four conserved cysteine residues that have two characteristic intramolecular disulphide bridges. According to the position of the two most amino-proximal cysteine residues, chemokines are distributed within two broad categories: the CXC chemokines in which the same two cysteine residues are separated by another amino acid and the C–C chemokines with two adjacent cysteines. In general, CXC chemokines Že.g. IL-8. attract neutrophils and

the C–C chemokines Že.g. RANTES, MCP-1 and MIP-1 b ., mononuclear leucocytes. Exceptions to this role and which are relevant to lymphocyte passage across the BBB are the CXC chemokines, IP-10, which is active for monocytes but not neutrophils and Mig, which is chemotactic for T lymphocytes. Chemokines bind avidly to the glycocalyx of endothelial cells and hence are appropriately positioned to activate leucocytes in close proximity. Examination of MS lesions by in situ hybridisation and immunohistochemical staining reveals RANTES, MCP-1, -2 and -3, MIP-1 a and IP-10 within infiltrating and resident cells w129–131x. Similar results pertain to EAE where CNS expression is directly related to clinical disease activity w132–135x. Antagonising the expression of MCP-1 w132x, MIP1 a w133x or IP-10 w134x by specific antibodies ameliorates the progression of EAE. Chemokine receptors are differentially expressed on Th1 and Th2 lymphocytes as demonstrated by the preferential association with Th1 of CXCR3 Žreceptor for IP-10 and Mig. and CCR5 Žreceptor for MIP-1 a , MIP-1 b and RANTES. and with Th2 of CCR3 Žreceptor for RANTES, MCP-3, MCP-4. w135x. In MS brain, the majority of mononuclear cell infiltrates are CXCR3 positive with CCR5-positive cells predominating in active demyelinating lesions w136x. Elevated levels of RANTES, MIP-1 a , IP-10 and Mig, whose activity is mainly directed at Th1 cells w137x, occur in the cerebrospinal fluid of MS patients which is coincident with an enrichment in the fluid of CD4q T cells bearing CXCR3 and CD4qand CD8q T cells bearing CCR5 w136x. Of the CSF chemokines, only MIP1 a and RANTES are associated with the enhanced migration of T lymphocytes from MS patients, an effect that is ascribed to the over-expression of CCR5 on these cells w138x. By appropriating different cytokines to the recruitment of T cell subclasses, their pattern of temporal expression could underlie variations in the stages of disease activity and progression w139x. The observation that chemokine expression in the CNS does not precede the onset of EAE w140x implies that these chemotactic factors are not involved in the early recruitment of antigen-specific T lymphocytes across the BBB but in the later mobilisation of non-specific inflammatory leucocytes. It appears that several chemokines are actively participating in the recruitment of lymphocytes to

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sites of cerebral inflammation. Whether modifying the activity of one of these cytokines rather than others will produce a greater inhibition of lymphocyte extravasation remains to be resolved. For the pragmatists in this area, it is of concern that the chemokine family has so many members, several of which possess overlapping functions. Should antagonising the expression of one chemokine lead to the over-expression of another with similar activity, then taking ‘pot-shots’ at leading members of the cast is likely to have limited therapeutic application if there is considerable redundancy of function within the chemokine family. Areas of work that have not been satisfactorily addressed include ascertaining whether some chemokines preferentially co-ordinate the trafficking of T lymphocytes that recognise cryptic myelin epitopes prior to disease relapse and whether the migration across the BBB is dependent upon the co-expression or multiple expression of a distinct array of chemokine receptors. Initial concerns of the complex interactions between the vast numbers of chemokines and of deciphering their pathological relevance may be overstated if further work enhances the notion that there is a sequential orchestration of chemokine activity that underlies the orderly recruitment of mononuclear leucocytes into the CNS. An additional illustration of this view is the suggestion that CD4q T lymphocytes influence the outcome of virus-induced demyelination by modifying the CNS expression of RANTES, which in turn regulates the migration of monocytes across the BBB w141x. Perhaps regulation of the migration of mononuclear leucocytes into the CNS is not a prerogative of one or a few key members of the chemokine family but is dependent upon the activities of a team whose strength lies in its close integration, durability and sophistication.

10. Matrix metalloproteinases Lymphocyte extravasation into the CNS includes penetrating the sub-endothelial basement membrane, which envelops the brain vessels. This and other obstacles are overcome by the activity of proteases such as matrix metalloproteinases ŽMMPs. which are comprised of at least 20 family members and which are efficient at degrading protein components of the

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extracellular matrix w142x. The contribution of MMPs to demyelination is adequately reviewed by Hartung and Kieser w143x. Potential sources of MMPs include resident CNS cells, endothelial cells and infiltrating leucocytes. Because of their destructive potential, the activity of the enzymes is controlled at the intracellular level by regulators of gene transcription and proenzyme activation and at the extracellular level by the formation of complexes with tissue inhibitors of MMPs ŽTIMPs.. An imbalance between TIMPs and MMPs could lead to disruption of the BBB w144x, enhanced secretion of TNFa w145x, degradation of myelin w146x and hence underlie the role of disease progression in MS w147,148x. In rat brain, MMP-2, MMP-7, MMP-8 and MMP-9 break down the extracellular matrix, the BBB, and increase leucocyte recruitment w149,150x. Induction of MMP-2 by VLA-4 facilitates T cell entry into the CNS during the early stages of EAE w81x. The MMPs gelatinase b , MMP-2, MMP-3, MMP-7 and MMP-9 are present in MS lesions and elevated levels detected in the CSF of MS patients w151–153x. Gelatinase b enhances the transbasement membrane migration of T lymphocytes w154x. Infiltrating T lymphocytes appear to be the main source of MMP-2 and MMP-9, though brain endothelial cells are also believed to make an active contribution to MMP-9 production w155x.

11. Concluding comments and therapeutic intervention This review has highlighted several stages at which factors may be operating to initiate and perpetuate the extravasation of lymphocytes across the BBB. In developing an effective strategy for combating lymphocyte penetration of cerebral vessels, there is a need to know which of these stages is most relevant to lymphocyte extravasation and is susceptible to selective antagonism that will lead to clinical benefit. From animal models, it is apparent that inhibiting the expression of adhesion molecules on blood lymphocytes or of cytokines and chemokines within CNS tissue significantly impedes lymphocytic infiltration and ameliorates CNS dysfunction. Such observations raise the question of whether some of the

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molecular and cellular features of EAE are exquisitely sensitive to pharmacological intervention, whether they have equitable pathological significance to MS and whether their antagonism will lead to successful therapeutic approaches in man. In extrapolating findings in EAE to MS, we must not lose sight of the fact that in EAE the initiating stimulus of CNS insult is known unlike MS whose aetiology remains one of conjecture. Also, disease modification by systemic intervention in EAE may be related to the high proportion of circulating mononuclear cells to polymorphonuclear cells in rodents, which is the inverse of that found in man. Other important differences between the animal models and MS is that EAE in rats, unlike that in some mice models, shows mononuclear cell infiltration without demyelination and the absence of a relapsing–remitting phase. Genetic heterogeneity exists among patients with MS, whereas EAE is induced in inbred rodent strains. The above reservations may dissipate with the development of EAE in the marmoset, which seems to provide a model whose clinicopathological features closely resemble MS w156,157x. In animal experiments, assessing the relative merits of a potential disease-modifying agent is often undertaken in deference to effects on other physiological parameters. For example, information is often found wanting concerning whether antagonising the expression of an adhesion molecule implicated in leucocyte extravasation into inflammatory sites modifies lymphocyte recirculation or impairs entry into sites of exogenous infection. Is antagonising the expression of one molecule likely to be of limited value in view of the redundancy of function that exists among chemokines, cytokines and possibly adhesion molecules and MMPs? The early description that inhibiting VLA-4 expression had a profound effect on the progression of disease activity in EAE produced a wave of optimism that here at last was a therapy that possessed the potential to have a considerable impact upon the demyelinating disorders w75x. Unfortunately, the early clinical trials of anti-VLA-4 antibodies in MS did not reach the level of expectation that was initially anticipated though this may have been related to their short-term duration w84x. Hopefully, a more rewarding outcome will emanate from longer trials that could also incorporate combination therapy.

Perhaps we should accept the pragmatic view that, despite the encouraging results gained from animal experiments, modifying the expression of one molecule is not going to significantly compromise lymphocyte extravasation since man’s immune system has evolved sufficiently to thwart any such eventuality by its development of a complicated series of back-up mechanisms. The demise of one factor may simply lead to the insertion of another into the inflammatory niche. Some molecules may be more prominent than others in controlling or unbalancing the homeostatic properties of the BBB. This point is illustrated by TNFa , which is proposed to occupy a pivotal role in the immunopathogenesis of rheumatoid arthritis w158x and that antagonising its activity will lead to a suppression of other pro-inflammatory cytokines. Recent anti-TNFa clinical trials in patients with rheumatoid arthritis have produced encouraging results w159x and inhibiting TNFa activity in MS would seem to be a reasonable therapeutic approach. However, the finding that administration of anti-TNFa antibodies to two MS patients increased disease activity w160x furthers the controversial claim that TNFa is, contrary to normal expectation, an anti-inflammatory cytokine in autoimmune-mediated demyelination w161x. One of the intransigent problems of disease intervention in MS is the penetration of the BBB by pharmacological antagonists of CNS targets. Pertinent to this strategy would be attempts to modify the expression of factors produced within brain lesions that recruit circulating lymphocytes either directly, e.g. chemokines, or indirectly by assisting passage though the various compartments of blood vessel walls Že.g. MMPs.. By its very nature, this approach will be highly specific but inherent in its design is the problem of exclusion by the BBB. At first sight, such concerns would seem to be unfounded since entry into the CNS should be facilitated by a damaged BBB. However, is the BBB truly damaged in MS and, if so, to what extent? From an early report, which suggested that the BBB was intact in 80% of patients with MS w162x, it is more likely that the barrier is compromised in MS rather than damaged. This again may have a significant bearing on the interpretation of data generated by EAE studies where disruption of the BBB may be more widespread than that seen in MS and where an increase in the electrical resistance

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of endothelial cells by phosphodisterase inhibitors stabilises the BBB w163x. An alternative approach is to design new therapeutic agents based upon drugs known to be of clinical benefit to MS patients and which impede lymphocyte migration into the CNS. Steroids, which for many years have been the mainstay treatment for MS, enhance TIMP levels and decrease the concentration of MMP-9 in the CSF of MS patients w164x. Such an effect is unlikely to be specifically directed at CNS pathology since steroids are known to be general inhibitors of cell-mediated immunity as illustrated by their suppression and release of cytokines and prostaglandins. More recently, treatment of MS patients with IFN b induced the progression of disease disability, in development of brain lesions and the relapse frequency w165–167x. The demonstration that interferon-b impedes T cell migration in vitro w168,169x, possibly through the secretion of gelatinase w170x or MMP-9 w171x, has fuelled the speculation that its mode of action includes antagonising the passage of leucocytes across the BBB. The drug inhibits mRNA expression of RANTES and MIP-1 a w172x, downregulates CCR5 w172x and VLA-4 expression of blood lymphocytes w173x, increases soluble VCAM-1 w174x and ICAM-1 w175x and decreases the number of leucocytes in CSF w176x. These reports do not mutually exclude other activities of IFN b , which include inhibiting IFNg synthesis, downregulating MHC Class II expression and restoring regulatory function in MS by increasing levels of the anti-inflammatory cytokine IL-10 w177x. A particular advantage of monitoring the behaviour and expression of immune components from therapeutically challenged MS patients is that studies are being performed on cells whose functional status has been modified by the underlying disease. Of relevance to this consideration is the enhanced migration of blood T lymphocytes from patients with MS w178x and their supranormal adhesion to TNFa-stimulated endothelial monolayers w71x. Where possible, it would be prudent for most in vitro studies, which involved the unravelling of T cell function in relation to MS, to be performed on cells isolated from patients with the disease. Because of ethical and practical restraints, it is not possible to undertake investigations on endothelial cells isolated from MS brain. Nevertheless, considerable benefit will still accrue from studying

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human or animal brain endothelial cells as their phenotypic and functional characteristics are likely to be quite distinct from non-CNS endothelial cells as illustrated by quantitative differences in the expression of MIP-1 b w179x, MMP-2 and MMP-9 w155x. At present, there does not appear to be a particular phase of lymphocyte entry across the BBB that is highly susceptible to pharmacological intervention. From a medical standpoint, preventing the initial CNS infiltration of antigen specific T lymphocyte will have little impact upon the treatment of patients who only present themselves at clinic as a consequence of the clinical features arising from CNS insult. Inducing T cell anergy with antibodies against co-stimulatory molecules or peptide ligands and using T cell receptor peptide vaccination to modify interactions between T cell receptors are procedures that change the clinical course of EAE. Applying such molecular manipulation to MS may not be so rewarding as shown by the disappointing outcome of recent trials of myelin-induced nasal tolerance. Critical to the success of any antigen-driven therapy in MS is defining not only the initial antigens that triggered the immune response but also those that may materialise from epitope spreading. Therefore, it is to be reasoned that any erstwhile therapeutic intervention operating at the level of the BBB needs to be directed at suppressing the CNS entry of circulating lymphocytes, prior to or during disease relapse. Should this approach take the form of antagonising a single factor or a combination that act in concert to generate the pathological cascade remains one of conjecture. On reflection, it would be desirable to inhibit contact of lymphocytes with cerebral walls for without attachment tissue extravasation will not ensue. Analogous to this view is the military stratagem that in preventing a sea-borne invasion, it is more rewarding to defeat the enemy before he is firmly entrenched on the shore. Once bound to endothelial cells of the compromised BBB, lymphocytes will enter the CNS parenchyma either in response to one of the many chemotactic factors produced by the inflammatory milieu or simply by random migration. Even this approach might be deemed as naıve ¨ since it does not address variations in the regulation of lymphocyte migration between cerebral vessels within discrete anatomical sites of

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the brain w180,181x and which specific subset of lymphocytes is to be targeted. Work in the author’s laboratory reveals that CD4q memory T lymphocytes differ from CD8q memory T lymphocytes in their expression of adhesion molecules and it is to be anticipated that this will also be true of Th1 and Th2 cells once markers are available to delineate these subsets. Preventing lymphocyte–endothelial interaction at the level of the BBB would be significantly aided by the identification of ligands restricted to cerebral endothelial cells and by characterisation of their corresponding adhesion receptors on lymphocytes. A successful outcome to this work would also have a significant bearing on the recruitment of circulating monocytes and dendritic cells whose persistence in the CNS is likely to maintain autoimmune reactivity and demyelination w182x. With guarded optimism, it is anticipated that the near future will see rapid inroads into the unravelling of the molecular features of lymphocyte–cerebral vessel wall interaction. The introduction into the clinic of specific antagonists of CNS inflammation is long overdue.

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