Leukocyte trafficking into the CNS

The CNS comprises of the brain and the spinal cord (SC), present within the cranial cavity and vertebral column, respectively. The CNS is protected by layers of fibro-collagenous tissue collectively called the meninges. Dura mater is the thicker outer most layer that lines the inside of the skull bones and consists of fibro-collagenous connective tissue and large blood vessels. The arachnoid and pia mater are thinner finer layers and are collectively called the leptomeninges. The pia mater is the innermost layer and lines the brain. The irregular space between the dura and pia is the cerebrospinal space. This CSF filled space is criss-crossed by a dense stroma and contains cerebral and spinal arteries (Ransohoff and Engelhardt, 2012).

Under normal homeostasis, the CNS is protected from a variety of cell populations and molecules that can potentially harm the brain. This property, for which the CNS is considered as immune-specialized site, is characterized by physiological and physical barriers such as: absence of lymphatics, lack of classical antigen-presenting cells (APCs), low expression of antigenic molecules (MHC-I and MHC-II) and the physical barriers between the circulation and the brain parenchyma. Under physiological circumstances, circulating cells do not enter the brain parenchyma, while only a small population of leukocytes is seen in the CSF. Nonetheless, leukocytes are capable of crossing the barriers and entering the CNS in cases of acute injury (e.g meningitis) or chronic insult (multiple sclerosis (MS)) (Ransohoff and Engelhardt, 2012, Romo-Gonzalez et al., 2012). Leukocyte are shown to enter the CNS across a number of routes described below and summarized in Figure 1-9.

1.6.3.1 Blood-brain barrier

The BBB is primarily formed by the brain microvascular endothelial cells (BMVECs) surrounded by basement membrane, pericytes and astrocytic foot processes. The BMVECs maintain the barrier function by the following features: Lack of fenestrations, relatively poor pinocytic ability, and presence of tight junctions. The barrier function is further reinforced by the surrounding basement membrane (Takeshita and Ransohoff, 2012). Moreover, the capillaries are guarded by the astrocytic foot processes, preventing the free

entry of leukocytes and macromolecules into the CNS. Astrocytes almost completely ensheath the brain vessels, separated only by the basement membrane (Hawkins and Davis, 2005). The pericytes cover a minor proportion of the capillaries and post capillary venules. These features almost completely seal the brain from large molecules, and immune cells. In this pathway, the cells are required for transition across the capillary endothelium, the basement membranes on the inner and outer siders of BMVECs to enter the parenchymal perivascular space. To reach the brain parenchyma, leukocytes are further required to cross the Glia Limitans (basement membrane and astrocytic foot processes) (Wilson et al., 2010). During infection, the inflamed endothelium upregulates selectins and adhesion molecules which, in turn, facilitates leukocyte rolling and adhesion (Wilson et al., 2010). In overt brain infections, leukocytes utilize matrix metalloproteinases (MMPs) to disrupt the glia limitans and enter the brain parenchyma (Rosenberg, 2002).

1.6.3.2 Blood-CSF barrier

As the name indicates, the BCSFB is the barrier between peripheral circulation and the CSF compartment. The BCFSB is located in the cerebral ventricles, and is composed of networks of capillaries embedded within a stroma underlying a monolayer of choroid plexus epithelial (CPE) cells. The capillaries of the BCSFB are fenestrated and contain gap junctions. Barrier function is maintained by tight junctions of the choroid plexus epithelium (CPE) cells (Man et al., 2007). The CPE cells generate CSF from the capillaries through diffusion and active transport. The CSF circulates in the CSF space and is re- absorbed through arachnoid villi in the cerebral venous sinuses. Cells migrating through this pathway are required to cross the fenestrated endothelium into the choroid plexus stroma and then cross the CPE to enter the CSF (Ransohoff and Engelhardt, 2012).

1.6.3.3 Virchow-Robin perivascular space

The vessels penetrating the CNS parenchyma are ensheathed by the leptomeninges for some distance giving rise to the Virchow-Robin perivascular space (Figure 1-9). Leukocytes may enter the cerebrospinal space while passing through the post capillary venules at the pial surface into Virchow-Robin perivascular surface (Man et al., 2007). It has been shown that the antigen presenting cells in this region are derived from circulation suggesting Virchow-Robin spaces are involved in cellular exchange between the circulation and the CNS (Hickey and Kimura, 1988).

Leukocyte populations in the CSF compartment are predominantly T lymphocyte subsets involved in routine immune surveillance. The median CSF leukocyte count in neonates and

young infants is 2-3/µl, however upto 20/µl can be normal (Kestenbaum et al., 2010) while in adults, the normal CSF leukocyte count is 1-3/µl. The majority (90%) of these cells are lymphocytes, mostly CD4+ memory T cells, 5% B-cells and monocytes each while infrequent dendritic cells (DCs) and polymorphonuclear cells are also seen (Kivisakk et al., 2003, Svenningsson et al., 1995, Seehusen et al., 2003). CCR7-CCL19 pair has been implicated in the entry of CCR7 expressing CD4+ central memory T (TCM) cells, (Kivisakk

et al., 2003) while CCL19 is constitutively present in the CSF (Krumbholz et al., 2007), Thus implying that constitutively present CCL19 at the BCSFB facilitates CCR7 expressing cells’ entry to the CSF compartment.

Leukocyte entry into the CNS has been studied extensively in experimental autoimmune encephalomyelitis (EAE) - a murine model of multiple sclerosis (MS). In this model, inflammation is initiated by myelin-specific T- lymphocytes routinely entering the CNS across the BCSFB where they are activated upon stimulation with myelin antigens. As a consequence, inflammatory signals from these ‘pioneer’ lymphocytes result in active recruitment of inflammatory cells into the brain parenchyma. The initial T-lymphocyte entry across the BCSFB rolling on the vascular surface is mediated by selectins (Sathiyanadan et al., 2014, Carrithers et al., 2000) following which the CCR6-CCL20 dependent interactions is important for leukocyte activation. In one such study, the T lymphocytes entered through the choroid plexus which expressed CCR6 ligand CCL20. Additionally, CCR6 KO mice were refractory to development of EAE (Reboldi et al., 2009). The activated lymphocytes utilize integrins such as α4β1 (Yednock et al., 1992) and VLA-4 (Carrithers et al., 2000) for adhesion and transmigration. CXCR3-CXCL10 interactions have also been reported in EAE with some conflicting results. For instance, using antibodies against CXCL10 were found to have a protective role of EAE (Fife et al., 2001). In contrast, another study showed that CXCR3 knockout mice were equally susceptible to EAE in comparison with CXCR3 wild-type mice (Liu et al., 2006). It can be conceived that the role for CXCR3 may be redundant if at all.

In summary, leukocyte subtypes transit across different compartments of the CNS during homeostasis and inflammation in an immune-privileged manner. There is some evidence to suggest that chemokine receptors may be important in controlling leukocyte migration to the CNS. The complexity of the CNS circulation and the redundancy leukocyte trafficking molecules present challenges in dissecting out the mechanisms of CNS entry of leukocytes.

Figure 1-9: Routes of leukocyte entry into the CNS

(a) A human head in the midline sagittal section showing relevant anatomical structures in schematic form. (b) arachnoid granulations in relation to the subarachnoid space and brain parenchyma. (c) Subpial space and Virchow-Robin space. (d) BBB and glia limitans. (e) Choroid plexus structure. Adapted from Ransohoff and Engelhardt (2012), Takeshita and Ransohoff (2012).