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Keywords:

  • adhesion molecules;
  • blood–brain barriers;
  • EAE;
  • T cell trafficking

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

Central nervous system (CNS) homeostasis is a prerequisite for the proper communication of neuronal cells. To this end, the endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier (BCSFB) tightly seal off the CNS from the continuously changing milieu within the blood stream. It is now well established that despite the presence of these barriers, the CNS is subject to immune surveillance and immune mediated pathogenic events. Numerous studies in an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), have elucidated that memory T cells can cross the non-inflamed BBB or BCSFB using specific molecular keys and gain access to the cerebrospinal fluid (CSF) drained ventricular, subarachnoidal and perivascular spaces. If these pioneer T cells encounter their specific antigen on antigen presenting cells strategically localized immediately behind the brain barriers, reactivation of the T cells will trigger a local inflammatory response, leading to the stimulation of the BBB. The activated BBB will then provide novel traffic signals allowing for the entry of large numbers of circulating inflammatory cells into the perivascular spaces and finally across the glia limitans into the CNS parenchyma, where they progress to initiate tissue injury. (Clin. Exp. Neuroimmunol. doi: 10.1111/j.1759-1961.2010.00009.x, May 2010)


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

Traditionally, the central nervous system (CNS) was viewed as an immunologically privileged site, which was interpreted as complete absence of immunosurveillance of this tissue.1 Theoretical considerations were that CNS homeostasis, which is required for the proper communication of neurons, would not tolerate routine immune cell patrolling on the search for relevant antigens. Experimental findings supporting this notion were that allo- and xenogeneic tissue grafts, when transplanted into the CNS, are much less efficiently rejected by the recipient as compared with orthotopic grafts. Additionally, the CNS parenchyma is devoid of constitutive expression of major histocompatibility complex (MHC) class I and II, and therefore the molecules required by T cells to recognize their antigen. Furthermore, lymphatic vessels, and thus the commonly established pathways of the afferent communication arm of the immune system, are also lacking. Last but not least, the efferent arm of the immune system to the CNS was considered blocked by the endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier (BCSFB), which efficiently protect the CNS from the constantly changing milieu within the blood stream.

This view of immunological ignorance of the CNS has, however, been in conflict with observations of Medawar et al., who suggested that a communication between the CNS and the immune system must exist.2 They showed that an allogeneic tissue graft into the brain, which would be tolerated in a naive host, was readily rejected by a recipient, who was sensitized to the allo-antigens before the transplantation. These observations suggested that T cells activated outside the CNS found a way across the brain-barriers and mounted an immune response within the CNS. Subsequent observations in a number of immune mediated CNS pathologies, including chronic inflammatory diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), made it obvious that the view of an immune privilege of the CNS as the absence of immune surveillance was in fact too extreme. We now know that T cells can indeed migrate across the BBB and the BCSFB, and if they recognize their antigen behind the barriers, they can mount (auto-) immune mediated pathogenesis within this tissue.3

T cells have to pass unique cellular barriers to enter the CNS

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

In order to access the CNS, circulating T cells have to succeed in breaching the unique cellular barriers in place to protect CNS homeostasis. The unique characteristics of the CNS microvascular endothelial cells comprise the lack of fenestrations, low pinocytotic activity, and highly complex and continuous tight junctions, reminiscent of those in epithelial cells.4 These BBB specific characteristics inhibit unspecific transcellular passage and paracellular diffusion of hydrophilic molecules across the BBB. At the same time, specific transport systems selectively expressed in the capillary endothelial cell membranes mediate the directed transport of nutrients into the CNS or of toxic metabolites out of the CNS to meet the high metabolic needs of the CNS tissue.5 While the BBB endothelial cells constitute the physical and metabolic barrier per se, proper barrier function is not intrinsically maintained by the endothelial cells alone but requires the continuous cross-talk with cellular and acellular components surrounding the CNS microvessels. In the fully differentiated BBB, the highly specialized endothelial cells are surrounded by an underlying basement membrane with a high number of embedded pericytes that are essential for vessel maturation and BBB development.6 The entire abluminal aspect of these parenchymal CNS microvessels is additionally ensheathed by a unique structure called the glia limitans perivascularis, consisting of a basement membrane with a composition of laminins that is distinct from that of the endothelial basement membrane7 and of a layer of astrocytic endfeet. Astrocytes play a major role in BBB maintenance by regulating the local water transport8 and by producing a number of growth factors relevant for barrier maturation and maintenance.9

Whereas at the level of CNS capillaries, the molecularly distinct basement membranes of the endothelial cells and of the glia limitans fuse to form one “gliovascular membrane”, they separate at the level of CNS post-capillary venules to seal off a cerebrospinal fluid drained perivascular space,10 in which the occasional antigen presenting cells can be found (Fig. 1).11,12 These perivascular spaces are open towards the subarachnoid space on the surface of the brain and spinal cord, facilitating drainage of extracellular fluids into the subarachnoidal space and thus compensating for the lack of lymph vessels in the CNS.13

image

Figure 1.  Multi-step recruitment of T cells across the central nervous system (CNS) parenchymal vessels. An overview of the adhesion and signaling steps involved in the multi-step T lymphocyte migration across the inflamed blood–brain barrier (BBB) during experimental autoimmune encephalomyelitis (EAE) at the level of CNS parenchymal microvessels. In the healthy CNS α4β1-integrin and VCAM-1 mediate the initial T cell capture to the BBB. It still remains to be shown if during ongoing EAE, P-selectin and its leukocyte ligand PSGL-1 and α4β1-integrin might be involved in lymphocyte tethering and rolling/capture. The initial contact is followed by G-protein dependent activation of α4β1-integrin and LFA-1, which mediate arrest of T cells to VCAM-1 and ICAM-1 on the endothelium, respectively. T cells will next crawl to sites permissive for diapedesis, with a probable involvement of endothelial ICAM-1 and ICAM-2. Finally, T cells might diapedese in a LFA-1/ICAM-1 and ICAM-2 dependent manner through the BBB endothelium, leaving tight junctions morphologically intact. Inflammatory T lymphocytes within perivascular spaces and the brain parenchyma during EAE can be characterized as effector/memory T cells with a characteristic CD45RBlowICAM-1highLFA-1highCD44highα4β1highα4β7low/−L-selectinlow/− surface phenotype. After penetrating the endothelial monolayer, T cells have to migrate across the endothelial basement membrane and encounter antigen-presenting cells in the cerebrospinal fluid drained perivascular space. Entry into the CNS parenchyma requires local digestion of the glia limitans perivascularis, composed of a second basement membrane and astrocytic endfeet.

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On the surface of the brain and spinal cord, the meningeal blood vessels lack the direct ensheathment by a glia limitans perivascularis.14 However, the CSF drained subarachnoid space is sealed off towards the neuropil by the glia limitans superficialis, which resembles the glia limitans perivascularis and surrounds the entire surface of the brain and spinal cord (Fig. 2).10 Subtle differences have been noted between parenchymal and meningeal microvessels, with parenchymal but not meningeal endothelial cells lacking storage of certain adhesion molecules in their Weibel–Palade bodies.15,16 Nevertheless, meningeal CNS microvessels also establish a functional BBB.17

image

Figure 2.  Multi-step recruitment of T cells across the central nervous system (CNS) meningeal vessels. An overview of the adhesion and signaling steps involved in the multi-step T lymphocyte migration across inflamed meningeal vessels during experimental autoimmune encephalomyelitis (EAE), as shown by intravital microscopy. At the onset of EAE, rolling of encephalitogenic T cells cannot be observed in meningeal microvessels and the molecular mechanisms operative during initial contact of the T cells at that time-point of the disease remain to be investigated. During ongoing disease, P-selectin and its leukocyte ligand PSGL-1, and to a lesser degree α4-integrin, mediate T cell tethering and rolling. This is followed by G-protein dependent activation of α4β1-integrin and LFA-1, which mediate arrest of T cells to VCAM-1 and ICAM-1 on the endothelium, respectively. T cells will next crawl in an α4β1-integrin and LFA-1 dependent manner against the direction of blood flow to sites permissive for diapedesis. Finally, T cells will diapedese in a LFA-1/ICAM-1 and ICAM-2 dependent manner through the BBB endothelium, leaving tight junctions morphologically intact. Inflammatory T lymphocytes present in the subarachnoid space during EAE are effector/memory T cells with a characteristic CD45RBlowICAM-1highLFA-1highCD44 highα4β1highα4β7low/−L-selectinlow/− surface phenotype. After penetrating the endothelial monolayer, T cells have to migrate across the endothelial basement membrane and encounter antigen-presenting cells in the CSF drained subarachnoid space. From there they can either migrate within perivascular spaces – the Virchow Robind spaces – deeper into the brain and reach parenchymal perivascular spaces or they directly traverse the glia limitans superficialis, composed of a second basement membrane and astrocytic endfeet. APC, antigen presenting cell.

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Besides the endothelial BBB, another major barrier sealing off the CNS from the changing milieu in the periphery is established by the epithelial cells of the choroid plexus, which form the BCSFB. In contrast to the endothelial BBB, until very recently this epithelial barrier has rarely been approached experimentally as a possible entry site for immune cells into the CNS.18–20 The choroid plexus extends from the ventricular surfaces into the lumen of the brain ventricles. Its major known function is the secretion of cerebrospinal fluid. It is a morphological structure organized in a villous surface, including an extensive microvascular network enclosed by a single layer of cuboidal epithelial cells.21 The microvessels within the choroid plexus parenchyma differ from those of the brain parenchyma, because they have fenestrations and intercellular gaps, and allow the free movement of molecules between the blood and the choroid plexus parenchyma.22 The barrier between the blood compartment and the CSF filled ventricles is located at the level of the choroid plexus epithelial cells, which form unique parallel tight junctions inhibiting paracellular diffusion of water soluble molecules (Fig. 3).18

image

Figure 3.  Multi-step recruitment of T cells across choroid plexus. Little is known about molecular mechanisms involved in T lymphocyte transit across the fenestrated choroid plexus microvessels and subsequent migration across the choroid plexus epithelium into the cerebrospinal fluid (CSF). In the uninflamed central nervous system (CNS), endothelial P-selectin mediates T cell 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, where de novo expression of MAdCAM-1 is also observed. Their polarized expression at the apical site of the blood–cerebrospinal fluid barrier (BCSFB) make them inaccessible for T cells in the choroid plexus stroma. Rather, CCL20 produced by choroid plexus epithelial cells mediated the migration of CCR6+ Th17 cells into the CSF filled ventricular space at the onset of experimental autoimmune encephalomyelitis (EAE). T cells in the CSF of healthy individuals are found to be mostly central-memory CD4+ T cells. Epiplexus cells are found on the surface of the BCSFB and might serve as antigen-presenting cells. APC, antigen presenting cell.

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T cell extravasation: A multi-step process with CNS specific steps added

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

The recruitment of circulating T cells into any given tissue has been shown to begin by the sequential interaction of different adhesion and/or signaling molecules on the T cell and the endothelial cells lining the vessel wall in a multi-step cascade.23 The multi-step interaction starts with an initial transient contact of the circulating immune cell with the vascular endothelium, mediated either by adhesion molecules of the selectin family and their respective carbohydrate ligands, or by α4-integrins and their ligands of the immunoglobulin (Ig) superfamily. After the initial tether, lymphoctes roll along the vascular wall with greatly reduced velocity. The rolling immune cell can then bind chemotactic factors of the family of chemokines presented on the endothelial surface. Chemokines bind to G-protein coupled serpentine receptors on the lymphocyte surface, delivering a G-protein-mediated inside-out signal to integrins present on the lymphocyte surface increasing their avidity by conformational changes and clustering. Activated integrins next mediate immune cell adhesion on the surface of the endothelium by binding to their endothelial ligands of the Ig superfamily. This ultimately leads to post-arrest crawling of the lymphocytes on the endothelium – mediated by integrins and their endothelial ligands – to a position on the endothelium that is permissive for the diapedesis of lymphocytes. Whereas adhesion molecules and chemokines mediating lymphocyte rolling and adhesion have been studied in detail, the molecular mechanisms involved in lymphocyte crawling on the endothelium and their diapedesis across the endothelium are in general less well understood. The same holds true for the molecular mechanisms involved in T cell migration through the endothelial basement membrane to access the tissue proper. Whereas peripheral tissues T cells would at this point have already entered the tissue parenchyma, in the CNS they at that point have just reached the CSF drained perivascular spaces, which are tightly sealed off from the CNS parenchyma by the glia limitans. Therefore, to access the CNS parenchyma, an additional barrier needs to be traversed by the immune cells. Considering the unique characteristics of brain endothelial cells and the unique architecture of the brain barriers, it is therefore plausible to hypothesize that similarly unique mechanisms apply for T cell entry into the healthy and inflamed CNS.

Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

Direct evidence for T cell entry into the healthy CNS was established by studying experimental autoimmune encephalomyelitis (EAE), an animal model for MS.24–26 EAE is a T cell mediated autoimmune disease, which can be induced either by immunization with CNS myelin antigens or by intravenous injection of freshly activated – but not resting – CNS myelin specific CD4+ T cell subsets into syngeneic naive recipients.27 In both EAE models, autoaggressive T cells are activated outside of the CNS and therefore must have the molecular keys to breach the brain barriers in order to access the CNS parenchyma and trigger inflammation, BBB breakdown and demyelination, which all set the stage for the development of the clinical manifestations of this disabling disease.

The first direct evidence that T lymphocytes can eventually cross the walls of parenchymal CNS microvessels in the absence of any overt inflammatory disease was obtained, when radioactively labelled encephalitogenic T cell blasts were traced after their intravenous injection into Lewis rats.28,29 Six hours after injection, very few of the T cells were found to have crossed the BBB and these cells could be detected in perivascular locations within the brain parenchyma.30 These studies also showed that a high activation-state rather than neuroantigen-specificity of the CD4+ T lymphoblasts was required for crossing the BBB, as resting T cells irrespective of their antigen-specificity could not be detected behind the BBB in these early studies.28,29

In agreement with these original findings, when directly observing the interaction of fluorescently labeled encephalitogenic T cell blasts in the spinal cord parenchymal microcirculation by intravital microscopy (IVM), our laboratory showed that freshly activated encephalitogenic T lymphoblasts can interact with the non-inflamed spinal cord white matter microvasculature.31 The recruitment of these T cells across the non-inflamed spinal cord white matter microvasculature was found to be unique as a result of the lack of rolling. Rather, encephalitogenic T cell blasts were observed to be promptly captured to the spinal cord white matter microvessel walls in an α4-integrin/VCAM-1 dependent manner. This was followed by a G-protein mediated increase in integrin avidity on the T cell surface in situ, which was required to allow for α4-integrin/VCAM-1 mediated firm adhesion to the vascular wall.31 The integrin leukocyte function associated antigen (LFA)-1 (αLβ2) was found to support post-adhesive T cell interaction with the spinal cord white matter microvascular wall, which seemed to follow specialized mechanisms, as successful diapedesis of T cells across the endothelial barrier could be first observed 4–6 h postinjection with T cells exclusively residing in perivascular localizations.32

Interestingly, very similar observations have been made when observing T cell interaction with non-inflamed retinal microvessels – which establish a blood–retina barrier comparable to the BBB – by using intravital scanning laser ophthalmoscopy.33 In the non-inflamed retina, activated T cells were observed to abruptly stop without prior rolling within retinal microvessels and to take 8–16 h to penetrate the blood–retinal barrier. Local activation of the endothelium as shown by the upregulated expression of ICAM-1 was shown to be a prerequisite of T cell diapedesis across this vascular barrier, as was the high activation state of the T cells, because resting T cells failed to interact with retinal microvessels in the absence of inflammation.33

Taken together, these observations show that highly activated but not resting T cells can eventually penetrate the parenchymal BBB in the absence of general CNS inflammation. Local T cell induced endothelial activation might, however, be a prerequisite for T cell diapedesis into the perivascular spaces, which would explain the extended time necessary for this process. Nevertheless, these studies showed that in the healthy CNS, T cell–endothelial cell encounters within the CNS microvessels are few and thus, it has been questioned whether T lymphoblast extravasation across parenchymal CNS microvessels would suffice for routine CNS immunosurveillance.

After the migration of fluorescently labeled encephalitogenic T cell blasts into the CNS, it was in fact shown that already at 2 h after peripheral injection T cells can be observed within the meninges and the choroid plexus parenchyma,34 suggesting that microvessels at these sites are easier to breach than CNS parenchymal microvessels. Interestingly, this study established a role for the adhesion molecule, P-selectin, in T cell recruitment across meningeal and choroid plexus microvessels. In the CNS, P-selectin is stored in the Weibel–Palade bodies of endothelial cells of meningeal vessels35 and the fenestrated choroid plexus capillaries19 but not in the Weibel–Palade bodies of CNS parenchymal vessels.35,36 Therefore the restricted expression of P-selectin in the endothelial cells of meningeal and choroid plexus microvessels might contribute to the different kinetics of T cell entry into selected sites within the CNS. In the end, P-selectin is, however, not required for T cell entry into the CNS, as functional blocking or absence of P-selectin does not influence the development of EAE.36–38

Therefore, although the traffic signals mediating T cell entry into the leptomeningeal spaces during immunosurveillance of the CNS still need to be defined, recent elegant two-photon IVM studies have confirmed the concept that T cell entry into the leptomeningeal spaces in the absence of inflammation is independent of the antigen-specificity of T cells.39 In this recent study, ovalbumin-specific T cells were observed to accumulate in significant numbers within pial blood vessels of Lewis rats 3 days after their peripheral injection. There they were observed to crawl for up to 10 min along the pial vessel wall, preferentially against blood flow before their diapedesis across the vascular wall. Once in the perivascular location, ovalbumin-specific T cells were not observed to leave the subarachnoid space to enter the CNS parenchyma, suggesting that in the absence of antigen-recognition on perivascular antigen presenting cells, the T cell fails to elicit the signals required for migration across the glia limitans.

T cells that have migrated across the non-inflamed meningeal microvessels have thus entered the CSF drained subarachnoid space and have therefore reached a location allowing immunosurveillance of the CNS to be carried out by screening the perivascular antigen presenting-cells. In contrast, T cells that crossed the choroid plexus microvasculature still resided within the choroid plexus parenchyma; that is, outside of the CNS. To access the CNS, they still need to breach the epithelial BCSFB. The presence of high numbers of central memory T cells in the cerebrospinal fluid (CSF), but not in the peripheral blood of healthy humans, implied that these T cells might enter the CSF space directly through the choroid plexus across the epithelial blood–cerebrospinal fluid barrier.19,40 In fact, the adhesion molecules, ICAM-1 and VCAM-1, are constitutively expressed on choroid plexus epithelium;41 however, with a polarized expression exclusively at the apical site of the epithelium,42 they are not accessible to T cells migrating across the BCSFB from the basolateral to apical site. The first molecular cues guiding T cell across the BCSFB could be identified recently, when studying the role of Th17 cells in EAE pathogenesis. It was observed that mice deficient for the chemokine receptor CCR6, which is expressed on Th17 cells, are resistant to the development of EAE.20 Careful examination of the CNS tissues of these mice after immunization with myelin-antigens showed not only the accumulation of CCR6-deficient immune cells in the choroid plexus parenchyma, but also that the CCR6 ligand, the chemokine CCL20, is constitutively expressed by choroid plexus epithelial cells. Combined with the observation that T cell interaction with the CNS microvessels is independent of CCR6, and EAE could be induced in CCR6-deficient mice by the transfer to a small number of myelin-specific CCR6 expressing Th17 cells, we concluded that CCL20 guides CCR6-expressing Th17 cells across the non-inflamed BCSFB. From there they can reach antigen-presenting cells localized on the surface of the choroid plexus epithelium or within the CSF drained perivascular or subarachnoidal spaces and trigger autoimmune CNS inflammation (Fig. 3).20 In apparent contrast to our study, others have merely observed a delayed onset of EAE in CCR6-deficient mice.43,44 We speculate that the immunization protocols used in these studies favor induction of Th1 over Th17 cells, which would readily enter the CNS through the endothelial BBB in a CCR6 independent manner. Finally, a recent study even observed increased severity of chronic EAE in CCR6-deficient mice,45 which might be a result of a lack of recruitment of CCR6+ regulatory T cells into the CNS at late stages of the disease as suggested before.43

The findings described earlier have established that immunosurveillance of the CNS does indeed take place. Under physiological conditions, the CNS strictly controls immune cell entry across its cellular barriers, which is exclusively restricted to activated effector/memory T cells. Without antigen-triggered activation, T cells apparently do not persist behind the brain barriers and also do not immigrate across the glia limitans into the CNS parenchyma proper.39,46 Thus, immunosurveillance of the CNS is strictly confined to these perivascular CNS compartments and does not impair the communication of the neuronal cells. If T cells, however, encounter their cognate antigen on perivascular antigen-presenting cells and are re-activated behind the brain barriers, they might then trigger an inflammatory response within this CNS compartment that leads to CNS autoimmune pathology.

Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

Reactivation of a T cell within the perivascular or subarachnoid spaces will activate the brain barriers leading to the expression of additional traffic signals on the BBB and the BCSFB, allowing the recruitment of additional inflammatory cells into the CNS. The molecular mechanism involved in T cell migration across the inflamed BBB have specifically been well studied and will be explained further.

Initial contact of T cells with the inflamed BBB

Visualizing the interaction of encephalitogenic T cells or endogenous circulating leukocytes with inflamed meningeal brain microvessels using IVM during ongoing EAE, a number of studies have shown a critical role for PSGL-1 and its endothelial ligand, P-selectin, in leukocytes and specifically CD4+ T cell rolling.38,47,48 In apparent contrast to these findings, CD8+ T cells, but not CD4+ T cells, from MS patients were found to use PSGL-1 to roll in inflamed meningeal brain venules of mice with EAE.49 Nevertheless, PSGL-1, or its endothelial ligands E- and P-selectin, seem to be dispensible for initiating T cell interaction with the inflamed CNS microvessels during EAE, as numerous observations by us and others showed that functional absence of PSGL-1 or its ligands E- and P-selectin have no significant impact on the recruitment of inflammatory cells into the CNS and thus the development of the clinical symptoms of EAE.36–38,50,51 Interestingly, the cytokine milieu, in which encephalitogenic T cells are activated, might influence the expression of functional PSGL-1. In our hands, Th1-driven encephalitogenic T cells showed poor binding to P-selectin, although they expressed high cell surface levels of PSGL-1 protein.51 To serve as a selectin-ligand, the protein backbone of PSGL-1 must be decorated with specific carbohydrate side chains. This can be influenced by interleukin-12, which was shown to enhance expression of functional PSGL-1 on myelin basic protein (MBP)-T cell receptor (TCR) transgenic (Tg)+ T cells during antigen-specific activation in vitro by inducing the enhanced transcription of C2GlcNAcT-I, which catalyzes the addition of branched O-glycan side chains to PSGL-1.52 IL-12 driven upregulation of functional PSGL-1 on these MBP-TCR tg+ T cells was found to correlate with their enhanced ability to transfer EAE into syngeneic recipients when transferring an unusually high number of MBP-TCR tg+ T cell blasts. In this specific scenario, the ability to transfer EAE was shown to depend on PSGL-1.52 As the presence of an increased number of circulating CD4+ T cells expressing high cell surface levels of P-selectin glycoprotein ligand (PSGL)-1 and showing an enhanced ability to interact with human brain endothelial cells in a PSGL-1-dependent manner in vitro has just been reported in MS patients,53 a possible involvement of PSGL-1 in T cell trafficking to the CNS needs to be further investigated.

In addition to PSGL-1, α4-integrins were reported to mediate rolling of CD4+ T cells and endogenous circulating inflammatory cells in inflamed meningeal vessels of the brain.48,49 However, functional blocking of α4-integrins on human T cells or absence of all β1-integrins, including α4β1-integrin, on mouse T cells failed to significantly reduce the initial interaction of these T cell populations with the inflamed spinal cord microvessels in mice with EAE.54,55 In contrast to blocking PSGL-1 and P-selectin, blockade of α4-integrin/VCAM-1 interaction inhibits the development of EAE in mice and the progression of MS in patients.56 Based on the finding that combined blockade of P-selectin and α4-integrins was more effective than inhibition of α4-integrins alone, it was suggested that both adhesive mechanisms might mediate T cell rolling in an overlapping or partially redundant fashion.38

Alternatively, at least during the onset of EAE, encephalitogenic T cells might adhere to the BBB without prior rolling as suggested by sophisticated two-photon imaging studies after the interaction of myelin-specific T cells with meningeal microvessels in the lumbar spinal cord of rats during the onset of clinical EAE.39 In this study, rolling encephalitogenic T cells could not be observed, suggesting that at this early time-point of the disease, encephalitogenic T cell blasts might still be promptly captured to the spinal cord meningeal microvessel walls as observed in the absence of CNS inflammation.

It therefore remains to be investigated if the activity of those traffic signals that mediate T cell rolling on the inflamed BBB is induced only at later time-points during EAE, switching the initial contact of T cells with the inflamed BBB from an α4-integrin mediated capture to a PSGL-1 mediated rolling. Alternatively, yet completely unknown traffic signals might be involved in initiating T cell interaction with the BBB at different time-points during EAE.

Induction of G-protein mediated signalling

The requirement for G-protein mediated signalling in T cell recruitment across the BBB during EAE has been shown by several studies,31,47 suggesting an involvement of chemokines or eicosanoids in inflammatory cell trafficking to the CNS during EAE. Many chemokines have been shown to be involved in the pathogenesis of EAE and have also been suggested to be involved in MS pathogenesis.57,58 Whether these chemokines are, however, truly involved in T cell trafficking across the BBB is largely unclear. Few chemokines have been described to be expressed by the BBB endothelium itself. These are the lymphoid chemokines CCL1959–61 and CCL21, which trigger adhesion of encephalitogenic CCR7+ T lymphocytes to inflamed brain vessels in Stamper–Woodruff assays in vitro.59 In this context, it is interesting to note that in the peripheral lymph node, PSGL-1 was recently shown to facilitate T cell trafficking across post-capillary venules by binding to CCL19 and CCL21.62

Another chemokine expressed in brain microvascular endothelium is CXCL12 and, although found to be upregulated during EAE, it might rather serve an anti-inflammatory role.63 CXCL12 is localized to the basolateral surface of CNS micovessels, where it retains perivascular CXCR4 expressing inflammatory cells within the perivascular space.63 In this study, blocking CXCR4 was shown to enhance inflammatory infiltration across the glia limitans into the CNS and enhance severity of EAE.

Most of the pro-inflammatory chemokines involved in EAE pathogeneis are produced by astrocytes and might either be released into the CNS parenchyma or into the CSF drained perivascular spaces. In order to mediate T cell interaction with the BBB, these chemokines must be transported from the abluminal to the luminal surface of the BBB endothelium.64 One of these chemokines is CCL2, which has been implicated in the pathogenesis of EAE. CCL2 can be transported through binding to its receptor, CCR2, in a caveolae-dependent manner from the abluminal to the luminal side of brain endothelium in an in vitro BBB model.65 Deficiency in CCR2 confers resistance to EAE and CCR2-deficient mice failed to develop mononuclear cell inflammatory infiltrates in the CNS and clinical EAE.66 A second study showed that CCR2-deficient encephalitogenic T cells are able to transfer EAE to wild-type mice, showing that at least T cell trafficking across the BBB does not require CCR2. It remains to be shown if other leukocyte subpopulations require CCL2 for migration into the CNS or if CCR2 might rather be involved in effector functions of inflammatory cells within the CNS.67

Thus, although these findings underline the importance of chemokines and their receptors in the pathogenesis of CNS inflammation, the chemokine(s) mediating the migration of circulating immune cells across the BBB in vivo remain to be identified. Having recognized that disease pathogenesis seems to start only once inflammatory cells have breached the glia limitans, it is conceivable that a number of the chemokines involved in EAE pathogenesis rather regulate T cell entry across this secondary barrier.

Adhesion of T cells to the inflamed BBB: T cell arrest and T cell crawling

During EAE, the adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are upregulated on meningeal and CNS parenchymal microvascular endothelial cells47,68–70 and interestingly also on the choroid plexus epithelium.41 The inflammatory cells localized within the CNS, stain positive for LFA-1 and α4β1-integrins, the ligands for ICAM-1 and VCAM-1, respectively, but not for L-selectin or α4β7-integrin.71–73 In MS lesions, upregulated expression of ICAM-1 is observed on inflamed vessels74,75 and most infiltrating cells stain positive for LFA-1.75 In contrast to the situation in EAE, VCAM-1 is found on CNS endothelial cells in some,76 but not other MS lesions.19,77 Interestingly, an alternatively spliced fibronectin isoform containing the CS1 region (FN-CS1) was suggested to serve as a potential α4-integrin ligand in human CNS microvessels.78

Numerous in vitro studies have provided evidence that T lymphocytes bind through LFA-1 and α4-integrins to their respective endothelial ligands, ICAM-1 and VCAM-1, on inflamed cerebral vessels in a Stamper–Woodruff assay69,79 or on cultured brain microvascular endothelial cells.78,80–83 Antibodies blocking α4-integrins or VCAM-1 were shown in a variety of animal models to prevent the development of EAE,70,72,79,84,85 and their therapeutic effect was attributed primarily to the inhibition of immune cell extravasation and inflammation in the CNS. Based on the important role of α4-integrins in leukocyte migration across the BBB observed in EAE, a humanized monoclonal anti-α4-integrin antibody Natalizumab was developed and has been successfully used for the treatment of relapsing-remitting MS.56 By means of intravital microscopy, we recently showed that Natalizumab specifically inhibits the adhesion, but not the rolling or capture, of human T cells within inflamed spinal cord microvessels in mice with acute EAE54 and thus provided the first direct in vivo proof of the concept of this therapy in MS. It should be noted that encephalitogenic T cells express two α4-integrins, namely α4β1 and α4β7. Although α4β7-integrin primarily binds to MAdCAM-1 expressed in mucosal tissues, on activated T cells it can also bind to VCAM-1 and could potentially be involved in T cell trafficking to the CNS.86 However, neutralizing α4β7-integrin antibodies failed to interfere with the development of EAE in SJL mice.72 Furthermore, β1-integrin deficient myelin-specific T cells fail to firmly adhere to inflamed spinal cord microvessels of mice with EAE,55 showing that α4β7-integrin cannot compensate for α4β1-integrin in mediating T cell adhesion to the inflamed BBB in vivo.

Despite its defined contribution in T cell adhesion to inflamed brain endothelium in vitro, functional inhibition or absence of LFA-1 or its endothelial ligand ICAM-1 in a variety of EAE models produced contradictory results, ranging from inhibiting EAE to increasing severity of EAE or having no effect at all,87–89 suggesting that further studies will be required to delineate the involvement of these molecules in EAE pathogenesis, also beyond T cell trafficking into the CNS.

After adhesion, T cells were shown to crawl on peripheral endothelium to sites permissive for diapedesis.23 While crawling on the endothelial surface, T cells have to resist detachment by the shear force rolling of CD4+ T cells they are exposed to within the bloodstream. This is achieved by the spatiotemporal regulation of the high and low-affinity forms of the α4-integrins and the β2-integrin LFA-1 along the axis of the crawling T cell.90–92

A recent two-photon analysis studying the interaction of myelin-specifc T cells with meningeal blood vessels during the onset of EAE provided the first direct in vivo evidence for post-arrest T cell crawling in inflamed meningeal microvessels.39 Interestingly, irrespective of their antigen-specificity, T cells preferentially crawled against the direction of the blood flow. This study further showed that infusion of blocking antibodies against α4-integrins, but not against LFA-1, significantly inhibited post-arrest T cell crawling along the vascular wall over time.39 In combination, however, these antibodies induced instantaneous detachment of myelin-specific T cells from the vascular wall, suggesting that although α4β1-integrin is dominant in mediating T cell arrest and crawling on the inflamed BBB, LFA-1 must have a minor or subordinate contribution to this adhesive interaction during the onset of EAE. The selective roles of α4β1-integrin/VCAM-1 and LFA-1/ICAM-1, and ICAM-2 interactions in T cell polarization and T cell crawling on the surface of brain endothelium preceding T cell diapedesis remain to be investigated.

T cell diapedesis across the inflamed BBB

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

To date, the sequence of molecular steps involved in diapedesis of T cells across the BBB are not well understood. In vitro studies under static conditions by us and others have shown that α4-integrin/VCAM-1 interactions are not involved in T cells diapedesis across brain endothelial cells,80 which is mediated by LFA-1/ICAM-1 and ICAM-2 interactions.93–95 Whereas the extracellular part of ICAM-1 suffices to engage T cell adhesion to brain endothelium, the cytoplasmic part of endothelial ICAM-1 is required for triggering T cell diapedesis involving RhoA activation in the brain endothelial cells.94 By using in vitro assays applying physiological shear, it has recently been shown that endothelial ICAM-1 mediates post-arrest T cell crawling and diapedesis on human umbilical vein endothelium.91 Similar in vitro studies have suggested the formation of specialized docking/capping structures between endothelial cells and lymphocytes. In particular, ICAM-1 and VCAM-1 form circular structures surrounding adherent lymphocytes, which are thought to facilitate the transmigration of T lymphocytes across the endothelial lining.96 An additional member of the Ig-superfamily, activated leukocyte cell adhesion molecule (ALCAM), was recently identified to localize to these docking structures on brain endothelium and to contribute to T cell diapedesis across the BBB in vitro through engagement of CD6 on CD4+ T cells.97 In this study, functional blocking of ALCAM was found to ameliorate EAE, showing an important function of this molecule in T cell migration across the BBB. Future studies therefore need to delineate the contribution of these adhesion molecules in mediating T cell polarization and crawling on the BBB versus their involvement in the final diapedesis step.

In contrast to the prevalent view that diapedesis of leukocytes occurs at and by separating the endothelial junctions, transmission electron microscopy on serial ultra-thin sections derived from brains of mice afflicted with EAE has shown only a few sites of neutrophil extravasation through tight junctions.98 In contrast, the majority of studies have shown that during EAE, inflammatory cell recruitment across the BBB leaves tight junctions morphologically intact,99 suggesting transcellular migration of immune cells across the BBB during EAE.100 The molecular mechanisms involved in transcellular diapedesis of immune cells across the BBB are not yet known. In addition, adhesion molecules localized to endothelial junctions, including CD99, CD99L, ESAM-1, L-1 and the JAM family members that have been implicated in extravasation of leukocyte subpopulations, have not been investigated with a specific focus on the BBB, with the exception of PECAM-1. PECAM-1-deficient mice presented with an earlier onset of EAE symptoms as a result of an increase in vascular permeability.101

T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

Having penetrated the activated CNS endothelial layer, infiltrating T cells next face additional obstacles on their way into the CNS parenchyma. A recent study suggested that the endothelial basement membrane constitutes an additional barrier for T cell penetration, allowing extravasation preferentially at sites containing laminin α4, but little or no laminin α5.7,102 In mice lacking laminin α4, there is compensatory ubiquitous expression of laminin α5 in endothelial cell basement membranes of the CNS. Interestingly, these mice were reported with ameliorated EAE as a result of the specific inhibition of T lymphocyte infiltration into the brain.102 These data suggest that laminin α5 in the basement membrane of the BBB does not support T cell migration across this basement membrane and rather directs the T cells to specific sites devoid of this laminin isoform. There, α6β1-integrin mediated binding of encephalitogenic T cells to laminin α4 will support their transmigration across the endothelial basement membrane.102

On penetration of the endothelial cell monolayer and its underlying basement membrane, extravasating T lymphocytes have reached the CSF drained perivascular space. To trigger EAE, encephalitogenic T cells have to re-encounter the cognate antigen in the context of MHC class II-bearing antigen-presenting cells in the CNS. A recent study has elegantly shown that antigen recognition on perivascular CD11c+ dendritic cells suffices to prime myelin-reactive T cells to trigger clinical disease development,12 which can only be seen once inflammatory cells penetrate the parenchymal basement membrane and glia limitans to enter he CNS parenchyma.103 In contrast to transmigration of the endothelial cell basement membrane, T lymphocyte penetration of the parenchymal basement membrane requires the activity of the matrix metalloproteinases, MMP-2 and MMP-9, which are apparently provided by myeloid cells rather than T cells recruited across the BBB.103 The reason why encephalitogenic T cells must use an alternative mechanism to penetrate the parenchymal basement membrane might be related to the different biochemical composition of these two basement membranes. It is important to note that encephalitogenic T cells cannot bind to the laminin isoforms present in the parenchymal basement membrane,7 suggesting that the glia limitans can only be breached by immune cells on its disintegration by extracellular matrix degrading enzymes during an inflammatory response mounted in the perivascular space.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References

Originally, the CNS was considered an immune privileged organ in the sense that it is ignored by the immune system. Recent studies on T cell trafficking to the CNS in the context of EAE have identified anatomical routes and molecular mechanisms of T cell entry into the CNS during health and disease, and suggest that CNS immune privilege can be compared with a medieval castle that is surrounded by a two-walled moat. Activated memory/effector, but not resting T cells, have specific molecular keys to traverse a small door through the intact outer wall – the BBB or the BCSFB – without impairing its resistance to assault. Behind this outer cellular barrier, T cells enter the CSF drained ventricular, perivascular and subarachnoidal spaces, which are bordered by two biochemically distinct basement membranes. Immunosurveillance of the CNS is confined to this space, which resembles the castle moat bordered by outer and inner walls, and patrolled by perivascular antigen presenting cells. If T cells encounter their specific antigen within the perivascular space, they become activated and will trigger an inflammatory response that leads to the upregulation of novel traffic signals on the BBB and the BCSFB; that is, opening doors in the outer wall and leading to the recruitment of additional inflammatory cells across this outer barrier. Production of cytokines and proteases allows the degradation of the glia limitans – the inner wall of the moat – and in a second step by lowering the drawbridge across the moat, a large number of infiltrating inflammatory cells will enter the CNS parenchyma, leading to the cellular assault of the castle, respectively. While our knowledge of the traffic signals involved in T cell migration across the BBB has grown enormously, the contributions of the BSCFB or the acellular barriers to T cell entry into the CNS to a great degree still remain to be investigated.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. T cells have to pass unique cellular barriers to enter the CNS
  5. T cell extravasation: A multi-step process with CNS specific steps added
  6. Experimental autoimmune encephalomyelitis as a model to study immune surveillance of the CNS
  7. Experimental autoimmune encephalomyelitis as a model to study inflammatory cell recruitment into the CNS
  8. T cell diapedesis across the inflamed BBB
  9. T cell migration into the CNS parenchyma: Additional acellular and cellular barriers to breach
  10. Conclusion
  11. Acknowledgements
  12. References
  • 1
    Barker CF, Billingham RE. Immunologically priviledged sites. Adv Immunol. 1977; 25: 154.
  • 2
    Medawar PB. Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue and to anterior chamber of the eye. Br J Exp Pathol. 1948; 29: 5869.
  • 3
    Engelhardt B, Ransohoff RM. The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol. 2005; 26: 48595.
  • 4
    Wolburg H, Wolburg-Buchholz K, Kraus J, Rascher-Eggstein G, Liebner S, Hamm S, et al. Localization of claudin-3 in tight junctions of the blood-brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multiforme. Acta Neuropathol (Berl). 2003; 105: 58692.
  • 5
    Pardridge WM. Blood-brain barrier delivery. Drug Discov Today. 2007; 12: 5461.
  • 6
    Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997; 277: 2425.
  • 7
    Sixt M, Engelhardt B, Pausch F, Hallmann R, Wendler O, Sorokin LM. Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis. J Cell Biol. 2001; 153: 93346.
  • 8
    Warth A, Kroger S, Wolburg H. Redistribution of aquaporin-4 in human glioblastoma correlates with loss of agrin immunoreactivity from brain capillary basal laminae. Acta Neuropathol (Berl). 2004; 107: 3118.
  • 9
    Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006; 7: 4153.
  • 10
    Owens T, Bechmann I, Engelhardt B. Perivascular spaces and the two steps to neuroinflammation. J Neuropathol Exp Neurol. 2008; 67: 111321.
  • 11
    Hickey WF, Kimura H. Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo. Science. 1988; 239: 2902.
  • 12
    Greter M, Heppner FL, Lemos MP, Odermatt BM, Goebels N, Laufer T, et al. Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis. Nat Med. 2005; 11: 32834. Epub 2005 Feb 2027.
  • 13
    Carare RO, Bernardes-Silva M, Newman TA, Page AM, Nicoll JA, Perry VH, et al. Solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: significance for cerebral amyloid angiopathy and neuroimmunology. Neuropathol Appl Neurobiol. 2008; 34: 13144.
  • 14
    Allt G, Lawrenson JG. Is the pial microvessel a good model for blood-brain barrier studies? Brain Res Brain Res Rev. 1997; 24: 6776.
  • 15
    Barkalow FJ, Goodman MJ, Gerritsen ME, Mayadas TN. Brain endothelium lack one of two pathways of P-selectin-mediated neutrophil adhesion. Blood. 1996; 88: 458593.
  • 16
    Yong T, Zheng MQ, Linthicum DS. Nicotine induces leukocyte rolling and adhesion in the cerebral microcirculation of the mouse. J Neuroimmunol. 1997; 80: 15864.
  • 17
    Bär T. The vascular system of the cerebral cortex. Adv Anat Embryol Cell Biol. 1980; 59: 162.
  • 18
    Engelhardt B, Wolburg-Buchholz K, Wolburg H. Involvement of the choroid plexus in central nervous system inflammation. Microsc Res Tech. 2001; 52: 11229.
  • 19
    Kivisakk P, Mahad DJ, Callahan MK, Trebst C, Tucky B, Wei T, et al. Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin. Proc Natl Acad Sci U S A. 2005; 100: 838994. Epub 2003 Jun 8326.
  • 20
    Reboldi A, Coisne C, Baumjohann D, Benvenuto F, Bottinelli D, Lira S, et al. C-C chemokine receptor 6-regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE. Nat Immunol. 2009; 10: 51423.
  • 21
    Dziegielewska KM, Ek J, Habgood MD, Saunders NR. Development of the choroid plexus. Microsc Res Tech. 2001; 52: 520.
  • 22
    Betz LA, Goldstein GW, Katzman R. Blood-brain-cerebrospinal fluid barriers. In Basic Neurochemistry: Molecular, Cellular, and Medical Aspects. SiegelGJ, ed. New York, NY: Raven Press; 1989: 591606.
  • 23
    Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol. 2007; 7: 67889.
  • 24
    Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol. 2005; 23: 683747.
  • 25
    Hohlfeld R. Multiple sclerosis: human model for EAE? Eur J Immunol. 2009; 39: 20369.
  • 26
    Gold R, Hartung HP, Toyka KV. Animal models for autoimmune demyelinating disorders of the nervous system. Mol Med Today. 2000; 6: 8891.
  • 27
    Ben-Nun A, Wekerle H, Cohen I. The rapid isolation of clonable antigen-specific T cell lines capable of mediating autoimmune encephalomyelitis. Eur J Immunol. 1981; 11: 195.
  • 28
    Hickey WF. Migration of hematogenous cells through the blood-brain barrier and the initiation of CNS inflammation. Brain Pathol. 1991; 1: 97105.
  • 29
    Wekerle H, Linington C, Lassmann H, Meyermann R. Cellular immune reactivity within the CNS. TINS. 1986; 9: 2717.
  • 30
    Hickey WF. Leukocyte traffic in the central nervous system: the participants and their roles. Semin Immunol. 1999; 11: 12537.
  • 31
    Vajkoczy P, Laschinger M, Engelhardt B. Alpha4-integrin-VCAM-1 binding mediates G protein-independent capture of encephalitogenic T cell blasts to CNS white matter microvessels. J Clin Invest. 2001; 108: 55765.
  • 32
    Laschinger M, Vajkoczy P, Engelhardt B. Encephalitogenic T cells use LFA-1 for transendothelial migration but not during capture and initial adhesion strengthening in healthy spinal cord microvessels in vivo. Eur J Immunol. 2002; 32: 3598606.
  • 33
    Xu H, Manivannan A, Liversidge J, Sharp PF, Forrester JV, Crane IJ. Requirements for passage of T lymphocytes across non-inflamed retinal microvessels. J Neuroimmunol. 2003; 142: 4757.
  • 34
    Carrithers MD, Visintin I, Kang SJ, Janeway CA Jr. Differential adhesion molecule requirements for immune surveillance and inflammatory recruiment. Brain. 2000; 123: 1092101.
  • 35
    Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD. Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell. 1993; 74: 54154.
  • 36
    Doring A, Wild M, Vestweber D, Deutsch U, Engelhardt B. E- and P-Selectin Are Not Required for the Development of Experimental Autoimmune Encephalomyelitis in C57BL/6 and SJL Mice. J Immunol. 2007; 179: 84709.
  • 37
    Engelhardt B, Vestweber D, Hallmann R, Schulz M. E- and P-selectin are not involved in the recruitment of inflammatory cells across the blood-brain barrier in experimental autoimmune encephalomyelitis. Blood. 1997; 90: 445972.
  • 38
    Kerfoot SM, Norman MU, Lapointe BM, Bonder CS, Zbytnuik L, Kubes P. Reevaluation of P-selectin and alpha 4 integrin as targets for the treatment of experimental autoimmune encephalomyelitis. J Immunol. 2006; 176: 622534.
  • 39
    Bartholomaus I, Kawakami N, Odoardi F, Schlager C, Miljkovic D, Ellwart JW, et al. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature. 2009; 462: 948.
  • 40
    Ransohoff RM, Kivisakk P, Kidd G. Three or more routes for leukocyte migration into the central nervous system. Nat Rev Immunol. 2003; 3: 56981.
  • 41
    Steffen BJ, Breier G, Butcher EC, Schulz M, Engelhardt B. ICAM-1, VCAM-1, and MAdCAM-1 are expressed on choroid plexus epithelium but not endothelium and mediate binding of lymphocytes in vitro. Am J Pathol. 1996; 148: 181938.
  • 42
    Wolburg K, Gerhardt H, Schulz M, Wolburg H, Engelhardt B. Ultrastructural localization of adhesion molecules in the healthy and inflamed choroid plexus of the mouse. Cell Tissue Res. 1999; 296: 25969.
  • 43
    Villares R, Cadenas V, Lozano M, Almonacid L, Zaballos A, Martinez AC, et al. CCR6 regulates EAE pathogenesis by controlling regulatory CD4+ T-cell recruitment to target tissues. Eur J Immunol. 2009; 39: 167181.
  • 44
    Liston A, Kohler RE, Townley S, Haylock-Jacobs S, Comerford I, Caon AC, et al. Inhibition of CCR6 function reduces the severity of experimental autoimmune encephalomyelitis via effects on the priming phase of the immune response. J Immunol. 2009; 182: 312130.
  • 45
    Elhofy A, Depaolo RW, Lira SA, Lukacs NW, Karpus WJ. Mice deficient for CCR6 fail to control chronic experimental autoimmune encephalomyelitis. J Neuroimmunol. 2009; 213: 919.
  • 46
    Flugel A, Berkowicz T, Ritter T, Labeur M, Jenne DE, Li Z, et al. Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis. Immunity. 2001; 14: 54760.
  • 47
    Piccio L, Rossi B, Scarpini E, Laudanna C, Giagulli C, Issekutz AC, et al. Molecular mechanisms involved in lymphocyte recruitment in inflamed brain microvessels: critical roles for P-selectin glycoprotein ligand-1 and heterotrimeric G(i)-linked receptors. J Immunol. 2002; 168: 19409.
  • 48
    Kerfoot S, Kubes P. Overlapping roles of P-selectin and alpha 4 integrin to recruit leukocytes to the central nervous system in experimental autoimmune encephalomyelitis. J Immunol. 2002; 169: 10006.
  • 49
    Battistini L, Piccio L, Rossi B, Bach S, Galgani S, Gasperini C, et al. CD8+ T cells from patients with acute multiple sclerosis display selective increase of adhesiveness in brain venules: a critical role for P-selectin glycoprotein ligand-1. Blood. 2003; 101: 477582.
  • 50
    Osmers I, Bullard DC, Barnum SR. PSGL-1 is not required for development of experimental autoimmune encephalomyelitis. J Neuroimmunol. 2005; 166: 1936.
  • 51
    Engelhardt B, Kempe B, Merfeld-Clauss S, Laschinger M, Furie B, Wild MK, et al. P-selectin glycoprotein ligand 1 is not required for the development of experimental autoimmune encephalomyelitis in SJL and C57BL/6 mice. J Immunol. 2005; 175: 126775.
  • 52
    Deshpande P, King IL, Segal BM. IL-12 driven upregulation of P-selectin ligand on myelin-specific T cells is a critical step in an animal model of autoimmune demyelination. J Neuroimmunol. 2006; 173: 3544.
  • 53
    Bahbouhi B, Berthelot L, Pettre S, Michel L, Wiertlewski S, Weksler B, et al. Peripheral blood CD4+ T lymphocytes from multiple sclerosis patients are characterized by higher PSGL-1 expression and transmigration capacity across a human blood-brain barrier-derived endothelial cell line. J Leukoc Biol. 2009; 86: 104963.
  • 54
    Coisne C, Mao W, Engelhardt B. Cutting edge: Natalizumab blocks adhesion but not initial contact of human T cells to the blood-brain barrier in vivo in an animal model of multiple sclerosis. J Immunol. 2009; 182: 590913.
  • 55
    Bauer M, Brakebusch C, Coisne C, Sixt M, Wekerle H, Engelhardt B, et al. {beta}1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity. Proc Natl Acad Sci U S A. 2009; 106: 19205.
  • 56
    Engelhardt B, Kappos L. Natalizumab: targeting alpha4-integrins in multiple sclerosis. Neurodegener Dis. 2008; 5: 1622.
  • 57
    Ubogu EE, Cossoy MB, Ransohoff RM. The expression and function of chemokines involved in CNS inflammation. Trends Pharmacol Sci. 2006; 27: 4855.
  • 58
    Ransohoff RM, Liu L, Cardona AE. Chemokines and chemokine receptors: multipurpose players in neuroinflammation. Int Rev Neurobiol. 2007; 82: 187204.
  • 59
    Alt C, Laschinger M, Engelhardt B. Functional expression of the lymphoid chemokines CCL19 (ELC) and CCL 21 (SLC) at the blood-brain barrier suggests their involvement in G-protein-dependent lymphocyte recruitment into the central nervous system during experimental autoimmune encephalomyelitis. Eur J Immunol. 2002; 32: 213344.
  • 60
    Columba-Cabezas S, Serafini B, Ambrosini E, Aloisi F. Lymphoid chemokines CCL19 and CCL21 are expressed in the central nervous system during experimental autoimmune encephalomyelitis: implications for the maintenance of chronic neuroinflammation. Brain Pathol. 2003; 13: 3851.
  • 61
    Krumbholz M, Theil D, Steinmeyer F, Cepok S, Hemmer B, Hofbauer M, et al. CCL19 is constitutively expressed in the CNS, up-regulated in neuroinflammation, active and also inactive multiple sclerosis lesions. J Neuroimmunol. 2007; 190: 729.
  • 62
    Veerman KM, Williams MJ, Uchimura K, Singer MS, Merzaban JS, Naus S, et al. Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs. Nat Immunol. 2007; 8: 5329.
  • 63
    McCandless EE, Wang Q, Woerner BM, Harper JM, Klein RS. CXCL12 limits inflammation by localizing mononuclear infiltrates to the perivascular space during experimental autoimmune encephalomyelitis. J Immunol. 2006; 177: 805364.
  • 64
    Dzenko KA, Andjelkovic AV, Kuziel WA, Pachter JS. The chemokine receptor CCR2 mediates the binding and internalization of monocyte chemoattractant protein-1 along brain microvessels. J Neurosci. 2001; 21: 921423.
  • 65
    Ge S, Song L, Serwanski DR, Kuziel WA, Pachter JS. Transcellular transport of CCL2 across brain microvascular endothelial cells. J Neurochem. 2008; 104: 121932.
  • 66
    Izikson L, Klein RS, Charo IF, Weiner HL, Luster AD. Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2. J Exp Med. 2000; 192: 107580.
  • 67
    Fife BT, Huffnagle GB, Kuziel WA, Karpus WJ. CC chemokine receptor 2 is critical for induction of experimental autoimmune encephalomyelitis. J Exp Med. 2000; 192: 899905.
  • 68
    Cross AH, Cannella B, Brosnan CF, Raine CS. Homing to central nervous system vasculature by antigen-specific lymphocytes. I. Localization of 14C-labeled cells during acute, chronic, and relapsing experimental allergic encephalomyelitis. Lab Invest. 1990; 63: 16270.
  • 69
    Steffen BJ, Butcher EC, Engelhardt B. Evidence for involvement of ICAM-1 and VCAM-1 in lymphocyte interaction with endothelium in experimental autoimmune encephalomyelitis in the central nervous system in the SJL/J mouse. Am J Pathol. 1994; 145: 189201.
  • 70
    Baron JL, Madri JA, Ruddle NH, Hashim G, Janeway CA Jr. Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma. J Exp Med. 1993; 177: 5768.
  • 71
    Engelhardt B, Laschinger M, Vajkoczy P. Molecular mechanisms involved in lymphocyte interaction with blood-spinal cord barrier endothelium in vivo. In The Blood-Spinal Cord and Brain Barriers in Health and Disease. SharmaHS, WestmanJ, eds. London: Academic Press; 2004: 1931.
  • 72
    Engelhardt B, Laschinger M, Schulz M, Samulowitz U, Vestweber D, Hoch G. The development of experimental autoimmune encephalomyelitis in the mouse requires alpha4-integrin but not alpha4beta7-integrin. J Clin Invest. 1998; 102: 2096105.
  • 73
    Engelhardt B, Martin-Simonet MT, Rott LS, Butcher EC, Michie SA. Adhesion molecule phenotype of T lymphocytes in inflamed CNS. J Neuroimmunol. 1998; 84: 92104.
  • 74
    Sobel RA, Mitchell ME, Fondren G. Intercellular Adheison Molecule-1 (ICAM-1) in cellular immune reactions in the human central nervous system. Am J Pathol. 1990; 136: 130916.
  • 75
    Bo L, Peterson JW, Mork S, Hoffman PA, Gallatin WM, Ransohoff RM, et al. Distribution of immunoglobulin superfamily members ICAM-1, -2, -3, and the beta 2 integrin LFA-1 in multiple sclerosis lesions. J Neuropathol Exp Neurol. 1996; 55: 106072.
  • 76
    Cannella B, Raine CS. The adhesion molecule and cytokine profile of multiple sclerosis lesions. Ann Neurol. 1995; 37: 42435.
  • 77
    Peterson JW, Bo L, Mork S, Chang A, Ransohoff RM, Trapp BD. VCAM-1-positive microglia target oligodendrocytes at the border of multiple sclerosis lesions. J Neuropathol Exp Neurol. 2002; 61: 53946.
  • 78
    Man S, Tucky B, Bagheri N, Li X, Kochar R, Ransohoff RM. Alpha4 Integrin/FN-CS1 mediated leukocyte adhesion to brain microvascular endothelial cells under flow conditions. J Neuroimmunol. 2009; 210: 929.
  • 79
    Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L, Karin N. Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature. 1992; 356: 636.
  • 80
    Laschinger M, Engelhardt B. Interaction of alpha4-integrin with VCAM-1 is involved in adhesion of encephalitogenic T cell blasts to brain endothelium but not in their transendothelial migration in vitro. J Neuroimmunol. 2000; 102: 3243.
  • 81
    Floris S, Ruuls SR, Wierinckx A, Van Der Pol SM, Dopp E, Van Der Meide PH, et al. Interferon-beta directly influences monocyte infiltration into the central nervous system. J Neuroimmunol. 2002; 127: 6979.
  • 82
    Greenwood J, Wang Y, Calder VL. Lymphocyte adhesion and transendothelial migration in the central nervous system: the role of LFA-1, ICAM-1, VLA-4 and VCAM-1. off. Immunology. 1995; 86: 40815.
  • 83
    Seguin R, Biernacki K, Rotondo RL, Prat A, Antel JP. Regulation and functional effects of monocyte migration across human brain-derived endothelial cells. J Neuropathol Exp Neurol. 2003; 62: 4129.
  • 84
    Kent SJ, Karlik SJ, Cannon C, Hines DK, Yednock TA, Fritz LC, et al. A monoclonal antibody to alpha 4 integrin suppresses and reverses active experimental allergic encephalomyelitis. J Neuroimmunol. 1995; 58: 110.
  • 85
    Keszthelyi E, Karlik S, Hyduk S, Rice GPA, Gordon G, Yednock T, et al. Evidence for a prolonged role of a4 integrin throughout active experimental allergic encephalomyelitis. Neurology. 1996; 47: 10539.
  • 86
    Ruegg C, Postigo AA, Sikorski EE, Butcher EC, Pytela R, Erle DJ. Role of integrin alpha 4 beta 7/alpha 4 beta P in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering. J Cell Biol. 1992; 117: 17989.
  • 87
    Archelos JJ, Jung S, Maurer M, Schmied M, Lassmann H, Tamatani T, et al. Inhibition of experimental autoimmune encephalomyelitis by an antibody to the intercellular adhesion molecule ICAM-1. Ann Neurol. 1993; 34: 14554.
  • 88
    Bullard DC, Hu X, Schoeb TR, Collins RG, Beaudet AL, Barnum SR. Intercellular adhesion molecule-1 expression is required on multiple cell types for the development of experimental autoimmune encephalomyelitis. J Immunol. 2007; 178: 8517.
  • 89
    Cannella B, Cross AH, Raine CS. Anti-adhesion molecule therapy in experimental autoimmune encephalomyelitis. J Neuroimmunol. 1993; 46: 4355.
  • 90
    Luster AD, Alon R, Von Andrian UH. Immune cell migration in inflammation: present and future therapeutic targets. Nat Immunol. 2005; 6: 118290.
  • 91
    Shulman Z, Shinder V, Klein E, Grabovsky V, Yeger O, Geron E, et al. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin. Immunity. 2009; 30: 38496.
  • 92
    Stanley P, Smith A, McDowall A, Nicol A, Zicha D, Hogg N. Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell. EMBO J. 2008; 27: 6275.
  • 93
    Reiss Y, Hoch G, Deutsch U, Engelhardt B. T cell interaction with ICAM-1-deficient endothelium in vitro: essential role for ICAM-1 and ICAM-2 in transendothelial migration of T cells. Eur J Immunol. 1998; 28: 308699.
  • 94
    Lyck R, Reiss Y, Gerwin N, Greenwood J, Adamson P, Engelhardt B. T-cell interaction with ICAM-1/ICAM-2 double-deficient brain endothelium in vitro: the cytoplasmic tail of endothelial ICAM-1 is necessary for transendothelial migration of T cells. Blood. 2003; 102: 367583.
  • 95
    Greenwood J, Amos CL, Walters CE, Couraud PO, Lyck R, Engelhardt B, et al. Intracellular domain of brain endothelial intercellular adhesion molecule-1 is essential for T lymphocyte-mediated signaling and migration. J Immunol. 2003; 171: 2099108.
  • 96
    Carman CV, Springer TA. Trans-cellular migration: cell-cell contacts get intimate. Curr Opin Cell Biol. 2008; 20: 53340.
  • 97
    Cayrol R, Wosik K, Berard JL, Dodelet-Devillers A, Ifergan I, Kebir H, et al. Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system. Nat Immunol. 2008; 9: 13745.
  • 98
    Cross AH, Raine CS. Central nervous system endothelial cell-polymorphonuclear cell interactions during autoimmune demyelination. Am J Pathol. 1991; 139: 14019.
  • 99
    Engelhardt B, Wolburg H. Mini-review: Transendothelial migration of leukocytes: through the front door or around the side of the house? Eur J Immunol. 2004; 34: 295563.
  • 100
    Wolburg H, Wolburg-Buchholz K, Engelhardt B. Diapedesis of mononuclear cells across cerebral venules during experimental autoimmune encephalomyelitis leaves tight junctions intact. Acta Neuropathol (Berl). 2005; 109: 18190.
  • 101
    Graesser D, Solowiej A, Bruckner M, Osterweil E, Juedes A, Davis S, et al. Altered vascular permeability and early onset of experimental autoimmune encephalomyelitis in PECAM-1-deficient mice. J Clin Invest. 2002; 109: 38392.
  • 102
    Wu C, Ivars F, Anderson P, Hallmann R, Vestweber D, Nilsson P, et al. Endothelial basement membrane laminin alpha5 selectively inhibits T lymphocyte extravasation into the brain. Nat Med. 2009; 15: 51927.
  • 103
    Agrawal S, Anderson P, Durbeej M, Van Rooijen N, Ivars F, Opdenakker G, et al. Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis. J Exp Med. 2006; 203: 100719.