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Regulation of Immune Cell Entry into the Central Nervous System

Britta Engelhardt1*
  • 1 University of Bern, Theodor Kocher Institute, Switzerland

Before entering the central nervous system (CNS) immune cells have to penetrate one of the brain barriers, namely the endothelial blood-brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB). Until recently the endothelial BBB has been regarded as the most obvious place for immune cell entry into the CNS and has therefore been investigated by most researchers. The BBB consists of a complex cellular system of highly specialized endothelial cells, a high number of pericytes embedded in the endothelial basement membrane, perivascular antigen-presenting cells, and a second basement membrane produced by astrocytes, which cover the abluminal aspect of the brain microvessels with their endfeet (summarized in (1). Although the endothelial cells form the BBB, i.e. the diffusion barrier, proper, alone they are insufficient to account for the unique barrier properties of CNS microvessels. Rather it is their interactions with extracellular matrix and cross-talk with adjacent cells that are pre-requisites for barrier function (2). These interactions in turn influence endothelial cell morphology, biochemistry and function that make BBB endothelial cells unique and distinguishable from any other endothelial cell in the body. Transcellular passage of molecules across the BBB is inhibited by an extremely low pinocytotic activity. The lack of fenestrae and an elaborate network of complex tight junctions (TJ) between the endothelial cells restrict the paracellular diffusion of hydrophilic molecules (3). Interestingly, although not ensheated by astrocytic endfeed barrier characteristics are also established in endothelial cells of meningeal CNS microvessels.
In contrast to the classical BBB, the epithelial blood-cerebrospinal fluid (CSF) barrier localized at the level of the choroid plexus epithelium, has until very recently not been considered as an entry site for immune cells or pathogens into the CNS (4). The choroid plexus extends from the ventricular surface into the lumen of the ventricles. Its major known function is the secretion of cerebrospinal fluid. It is a structure organized in a villous surface including an extensive fenestrated microvascular network enclosed by a single layer of cuboidal choroid plexus epithelial cells, which form the BCSFB.

It has been well established that successful recruitment of circulating leukocytes into a tissue depends on a multistep cascade involving the productive leukocyte/endothelial interaction during each of these sequential steps (5). As endothelial cells participate actively in the regulation of leukocyte entry into various tissues, it can be concluded that the specialization of the BBB endothelium extends to CNSspecific traffic signals for immune cells.

In order to define the multistep sequence of traffic signals involved in the recruitment of immunocompetent cells across the BBB, we have investigated the expression of adhesion molecules on BBB endothelium during experimental autoimmune encephalomyelitis (EAE) in the mouse as model for multiple sclerosis by means of in situ hybridization and immunohistology and investigated their functional involvement by performing in vitro adhesion and transmigration assays. Monoclonal antibody inhibition studies and adhesion molecule deficient mice were used to study the involvement of the respective adhesion molecules in EAE pathogenesis. Last but not least we have pioneered the use of intravital fluorescence videomicroscopy (IVM) in the spinal cord of mice to obtain direct in vivo evidence for the molecular mechanisms involved in immune cell trafficking to directly and noninvasively study the dynamics of immune cell/BBB interaction in vivo and analyse the consequences of experimental interference with individual traffic signals during EAE in vivo.

We have found that during EAE, α4β1-integrin/VCAM-1 are critically involved in leukocyte interaction with the inflamed BBB. Intravital microscopy studies of the spinal cord microvasculature of mice suffering from EAE have shown that blocking α4-integrin or lack of β1-integrin on myelin specific T cells does not allow them to maintain stable adhesions with the CNS microvascular endothelium and thus prohibits their migration across the BBB resulting in inhibition of EAE (6, 7). This α4β1-integrinmediated firm arrest to the inflamed BBB depends on heterotrimeric G(i)-linked receptors, however, the chemokines and chemokine receptors mediating leukocyte integrin activation – a prerequisite for firm adhesion - at the BBB are not well defined. The molecules mediating tethering and rolling of leukocytes to the inflamed BBB during EAE are presently controversially discussed. Although intravital microscopy studies of other laboratories have demonstrated the involvement of P-selectin and its ligand PSLG-1 in immune cell rolling in inflamed meningeal brain venules during EAE (8, 9), blocking these adhesion molecules or their entire absence in gene targeted mice fails to show any impact on EAE pathogenesis(10, 11), supporting the view that these molecules are dispensable for leukocyte interaction with the inflamed BBB during EAE. Similarly, blocking of α4-integrins or lack of β1−integrins on myelin specific T cells also fails to significantly impair initial capturing and rolling of T cell on the inflamed BBB (6, 7). Thus, it remains to be shown which adhesion receptors mediate this initial step of T cell interaction with the inflamed BBB during EAE in vivo.
Furthermore, the adhesion molecules involved in leukocyte diapedesis across the inflamed CNS microvessels during EAE still remain to be determined. In vitro we have found a critical role for endothelial ICAM-1 and ICAM-2 for T cell diapedesis across the BBB under static conditions (12). Using live cell imaging techniques we have recently dissected the individual contributions of endothelial ICAM-1 and ICAM- 2 on T cell interaction with the BBB under the influence of physiological flow and found a sequential involvement of endothelial ICAM-1 and VCAM-1 in mediating shear resistant T cell arrest followed by endothelial ICAM-1 and ICAM-2 in mediating T cell polarization and directed crawling to permissive sites of diapedesis on BBB endothelium.
Last but not least, the involvement of endothelial tight junctions in leukocyte recruitment across the BBB during EAE remains to be understood. During EAE, we reported a selective loss of claudin-3 immunostaining specifically from tight junctions of venules surrounded by inflammatory cuffs, whereas the localization of the other tight junction proteins remained unchanged (13). These findings implicated an involvement of endothelial tight junctions in leukocyte recruitment across the BBB. On the other hand we found that leukocyte diapedesis across the BBB during EAE leaves TJ morphologically intact (14). Using a transgenic mouse model that allows for TET-inducible and endothelial cell-specific expression of the TJ inducing protein claudin-1, which is absent in TJ of CNS parenchymal microvessels we therefore compared the development of EAE in double transgenic claudin-1- expressing C57Bl/6 mice to single transgenic control mice. Our study demonstrated that TET-induced claudin-1 did not affect inflammatory cell recruitment across the BBB during EAE, however, by reducing BBB leakiness ameliorated chronic EAE. These findings further support the notion that during EAE diapedesis of inflammatory cells across the BBB takes place via a transcellular route.

Whereas we begin to understand the molecular mechanisms mediating the multi-step entry of immune cell entry across the inflamed BBB during EAE much less is known about the molecular mechanisms involved in immune cell entry into the healthy CNS during immunosurveillance. By means of IVM we found that α4−integrins play a predominant role in mediating the passage of encephalitogenic Th1 blasts across non-inflamed spinal cord microvessels (15). In contrast Th17 cells were found to rather enter the healthy CNS via the BCSFB in a CCR6-CCL20 dependent manner (16), demonstrating different anatomical routes and molecular mechanisms involved in the immunosurveillance of the CNS.
Our future studies will therefore continue to delineate the different anatomical routes and molecular mechanisms involved in the entry of different immune cell populations into the CNS during immunosurveillance and disease.

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References

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7. M. Bauer, C. Brakebusch, C. Coisne, M. Sixt, H. Wekerle, B. Engelhardt and R. Fassler: {beta}1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity. Proc Natl Acad Sci U S A, 106, 1920-1925 (2009)

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12. R. Lyck, Y. Reiss, N. Gerwin, J. Greenwood, P. Adamson and B. Engelhardt: 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, 102(10), 3675-83 (2003)

13. H. Wolburg, K. Wolburg-Buchholz, J. Kraus, G. Rascher-Eggstein, S. Liebner, S. Hamm, F. Duffner, E. H. Grote, W. Risau and B. Engelhardt: 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), 105(6), 586-92 (2003)

14. H. Wolburg, K. Wolburg-Buchholz and B. Engelhardt: Diapedesis of mononuclear cells across cerebral venules during experimental autoimmune encephalomyelitis leaves tight junctions intact. Acta Neuropathol (Berl), 109(2), 181-90 (2005)

15. P. Vajkoczy, M. Laschinger and B. Engelhardt: Alpha4-integrin-VCAM-1 binding mediates G proteinindependent capture of encephalitogenic T cell blasts to CNS white matter microvessels. J Clin Invest, 108(4), 557-65 (2001)

16. A. Reboldi, C. Coisne, D. Baumjohann, F. Benvenuto, D. Bottinelli, S. Lira, A. Uccelli, A. Lanzavecchia, B. Engelhardt and F. Sallusto: 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, 10(5), 514-23 (2009) doi:ni.1716 [pii] 10.1038/ni.1716

Conference: Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU, Brussels, Belgium, 11 Feb - 12 Feb, 2010.

Presentation Type: Oral Presentation

Topic: Presentations

Citation: Engelhardt B (2010). Regulation of Immune Cell Entry into the Central Nervous System. Front. Pharmacol. Conference Abstract: Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU. doi: 10.3389/conf.fphar.2010.02.00008

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Received: 23 Feb 2010; Published Online: 23 Feb 2010.

* Correspondence: Britta Engelhardt, University of Bern, Theodor Kocher Institute, Bern, Switzerland, britta.engelhardt@tki.unibe.ch