Trafficking of immune cells in the central nervous system (original) (raw)
Adhesion molecules. Several studies that directly visualized the molecular steps that mediate the access of T cells to various compartments associated with the CNS have been performed by imaging the superficial vessels in the meninges that are associated with the spinal cord as well as pial vessels and parenchymal branches in a variety of model systems (74–78). In the context of EAE, several reports have described the initial tethering and rolling of leukocytes along the endothelium prior to firm adhesion (25, 79) and migration against the blood flow (74). Similarly, intravital microscopy studies of HSV-infected mice have demonstrated increased leukocyte rolling and adhesion in the microvasculature of the pia mater of infected mice (80), and similar neutrophil behaviors in the meninges have been identified during LCMV infection (64). These observations are consistent with the idea that inflammation in the CNS, either due to autoimmune responses or infection, leads to increased expression of adhesion molecules on endothelial cells of the BBB and choroid plexus, including members of the selectin family; cell adhesion molecules of the immunoglobulin superfamily, for example, ICAM1, VCAM1, and PECAM1; and members of the integrin family (Figure 3) (51, 53, 75, 81–83). This topic has been reviewed extensively elsewhere (41, 49, 76) and therefore is not discussed in detail here. Nevertheless, the biology of the integrin dimer VLA-4 (α4β1 integrin) and its ligand VCAM1, as well as their association with the development of MS and EAE, are particularly instructive in thinking about the need to balance immune access to the brain.
Leukocyte trafficking across the glia limitans into the parenchyma of the brain. Activated leukocytes expressing adhesion molecules and integrins roll and attach to the vascular endothelium. Successful diapedesis requires appropriate ligation of adhesion molecules, selectins, and integrins, signaling to both the infiltrating leukocyte and the brain endothelium. Expression of CXCL12 on the basolateral surface of endothelial cells recruits CXCR4+ T cells. However, retention of cells in the perivascular space occurs in the presence of high concentrations of CXCL10. Continued migration puts cells in contact with the glia limitans, which is composed of a highly structured wall of astrocytes. Further positive migratory signals, including chemokines, from these and surrounding cells may allow leukocyte migration into the parenchyma.
Although it is controversial as to whether VCAM1 is expressed in human vasculature, the finding that blockade of VLA-4/VCAM1 interactions delayed the onset and/or decreased the severity of EAE implicated this molecule as a target for the treatment of MS (84). This led to the clinical development of a monoclonal antibody (known as natalizumab) that targets α4 integrin (a component of VLA-4); natalizumab was successfully used in clinical trials to manage this condition (85). However, a small number of patients treated with this reagent developed progressive multifocal leukoencephalopathy associated with the reactivation of latent JC polyomavirus infection (20). This observation has been paralleled by the recent withdrawal of an antibody (known as efalizumab) that blocks LFA-1, which was used for the treatment of psoriasis and also led to the reactivation of JC polyomavirus in the brain (86). Whether JC polyomavirus persists in a latent form in the CNS or these events are a consequence of reactivation of the virus in peripheral tissues and subsequent spread to the CNS is unclear (87). However, recent reports that detected the presence of JCV in normal brain tissue support the former notion (88–90). Regardless, blockade of α4 integrin has also been shown to antagonize protective immune responses to multiple pathogens in the brain, including T. gondii (28), simian immunodeficiency virus–induced AIDS encephalitis (91), and Borna virus–induced progressive encephalitis (92). Other promising strategies to block cell trafficking to the CNS (77) may encounter similar problems. Despite all of these potential complications, the number of adverse events associated with natalizumab therapy has been lower than might have been predicted from studies in experimental systems, and this therapy continues to be used for the treatment of MS. Importantly, recent studies involving the generation of bone marrow–chimeric mice in which the hematopoietic compartment lacked β1-integrin but retained α4β7 integrin signaling indicated that T cell accumulation in the CNS during EAE required α4β1-integrin but that migration of granulocytes and macrophages into the CNS was independent of β1 integrin (93). These findings highlight the potential to control the trafficking of specific immune populations and perhaps even subsets of lymphocytes into the brain.
Astrocytes and immune cell trafficking in the parenchyma. Astrocytes provide an important structural component of the BBB (94) and are thought to restrict access of immune cells to the CNS. So, after rolling along, adhering to, and finally crossing the endothelial cells of the BBB and their associated basement membrane, the migrating leukocytes reach their next barrier, the glia limitans (Figure 3). This structure surrounds the blood vessel, is composed of astrocytic foot processes, is linked to the basal membrane by the transmembrane receptor dystroglycan, and forms its own molecularly distinct basement membrane composed of laminin, fibronectin, and type IV collagen (95, 96). There are very few studies that have considered how leukocytes cross this second barrier, although several reports have provided evidence that production of MMPs by immune cells is required for cleavage of dystroglycan and the breakdown of BBB during EAE and neurocysticercosis associated with tapeworm infection (96, 97).
Reactive astrocytes are a hallmark of most inflammatory responses in the brain and this activation corresponds with increased astrocyte numbers, changes in their morphology, and upregulated expression of glial fibrillary acidic protein, an astrocyte-specific structural protein (98). The seminal studies by Fontana et al. (99), which suggested that astrocytes could present antigen to CD4+ T cells, highlighted the possible role of these glial cells in the immune response. Although the ability of astrocytes to present antigen through MHC class II remains controversial (100), the formation of synapses between astrocytes and antigen-specific CD8+ T cells in vivo is consistent with their ability to present antigen through MHC class I (101, 102). Nevertheless, reactive astrocytes are frequently associated with migrating T cells and act as a source of multiple cytokines and chemokines during inflammation (103), which may actively promote cell trafficking into and within the CNS. However, in the majority of experimental systems it has not yet been defined how these interactions affect the coordination of antimicrobial immune responses.
The possible contribution of astrocytes to immune responses within the brain has been described in several settings, including those involving the targeted overexpression of cytokines — such as TNF, IFN-α, TGF-β, IL-6, and IL-12 — by astrocytes, which leads to chronic inflammation and progressive neurodegeneration (104–108). More recent studies analyzing mice in which the ability of astrocytes to participate in immune function is compromised through the specific loss of a cytokine receptor such as gp130 or reduced NF-κB signaling, have shown that this alters the course of immune responses in the CNS (109–112). Thus, in a mouse model of spinal cord injury, astrocyte-specific inhibition of NF-κB (which is necessary for the activation of many cytokine genes) resulted in a reduction in the number of reactive astrocytes in the CNS, in lower levels of chemokines, and in reduced infiltration of T cells and macrophages (111). Consequently, this led to improved spinal cord healing. Future challenges include determining how individual cytokines, adhesion molecules, and chemokines produced by astrocytes influence the development of inflammation and the behavior of infiltrating immune cell populations.
Chemokines and migration in the parenchyma. Once cells have crossed all the membrane barriers and gained access to the parenchyma of the brain, what molecular cues guide their migration? There is an extensive list of chemokines that are either expressed constitutively or upregulated in the brain during inflammation, and infiltrating immune cells express a wide array of chemokine receptors associated with chemotaxis and/or effector function. The use of mice lacking specific chemokines or chemokine receptors and treatment with antagonists of these interactions has provided useful insights into which interactions are likely important in the brain. However, one of the frequently raised caveats is that this may not distinguish between their role in the development of immunity versus trafficking of cells to the CNS (compare conclusions of refs. 29 and 57). For example, the increased susceptibility of mice that lack CCL3 to viral infection in the brain may be due to poor activation and priming of dendritic cells rather than to a failure of T cells to traffic to and migrate within the CNS (113).
Regardless, the relevance of chemokines to immune cells in the CNS remains an area of active research and has been covered extensively in other articles (76, 114), and therefore we only provide a summary of their role in various model systems (Table 2). However, it is helpful to highlight the range of pathologies in which these molecules appear to have critical roles. The chemokine receptors CCR2 and CCR5, which are expressed on many monocytes and T cells and, despite difficulties in detecting these receptors, in MS lesions (115–117), have been implicated in CNS inflammation because blockade of their interactions leads to a reduction in inflammation in mouse models of immune-mediated demyelination (118–123). These findings are broadly consistent with the ability of chemokines to mediate their activities through chemotaxis and activation of integrins (124), but they can also have more complex effects on cell behavior. The majority of T cells found in the uninflamed CNS express CXCR3 (59), and this receptor has been implicated in cerebral malaria pathology (125, 126) and recruitment of protective CTLs during viral infection (113, 127). Furthermore, the recruitment of CXCR3+ T cells to neurons infected with West Nile virus has been attributed to the localized production of CXCL10 by the infected cells (128). However, the role of CXCR3 and one of its ligands, CXCL10, during EAE appears more complex (60). During this autoimmune condition, expression of CXCR3, rather than inducing a chemotactic response, is implicated in the retention of autoimmune cells and Tregs in the perivascular space (60). It is also implicated in the retention of antiviral CD8+ T cells during LCMV infection (129). Similarly, CXCL12, the ligand for CXCR4, is constitutively expressed in the CNS on the basolateral surface of endothelial cells and is upregulated during neuronal inflammation, and the absence of CXCR4 signaling during EAE leads to perivascular accumulation of mononuclear cells in the spinal cord (130). These studies suggest that multiple chemokines regulate access from the perivascular space to the parenchyma.
Chemokines and their receptors implicated in the trafficking of immune cells into the CNS
Kinetics and behavioral analysis of lymphocytes within the brain parenchyma. Related to the themes of this review, CCR7 has an important role for T cell and dendritic cell recruitment to the lymph node, where its ligands CCL19 and CCL21 provide motogenic signals required for efficient T cell and dendritic cell migration (131, 132). It has been suggested that expression of CCL19 in the uninflamed parenchyma has a role in immune surveillance by CCR7+CD4+ memory T cells (56, 133), but this expression is elevated in MS lesions (56). CCL21 is also upregulated in other models of CNS inflammation (28), and whether these chemokines also provide motogenic signals in the parenchyma of the brain is unknown. Multiphoton microscopy of the spinal cord has been used to image the behavior of encephalitogenic cells within the white and grey matter during the induction of EAE (25, 74). A recent detailed study has pinpointed three distinct phases for encephalitogenic CD4+ T cell entry into the brain: (a) arresting to leptomeningeal vessels and scanning of the luminal surface against the blood flow; (b) diapedesis and scanning of the pial membrane for antigen being presented by perivascular macrophages; and (c) successful antigen-dependent activation of T cells, triggering effector capacity and resulting in tissue invasion (74). This study challenges the notion that the choroid plexus is the major route of cell entry during EAE and solidifies data suggesting an antigen-dependent mechanism. Following activation, during the initial disease process, myelin-specific cells enter the CNS in a rapid wave and can migrate deep into the parenchyma (134). These cells could be divided into two main populations based on migratory velocities (ranging from 6–25 μm/min). After entry into the perivascular space, many of the cells displayed a restrained or stationary phenotype, suggesting that they were forming long-term contacts with resident cells. This type of arrested behavior is associated with MHC/TCR interactions, although chemokines have also been implicated in mediating cell-cell interactions (135, 136). Nevertheless, transfer of myelin-specific (encephalitogenic) CD4+ T cells led to substantially more stationary cells in the brain than did the transfer of T cells not specific for CNS proteins (25). Somewhat unexpectedly, these studies revealed that T cell migration in this microenvironment was, at the population level, random, indicating that local migration was not regulated by chemokine gradients. Thus, although the arrest of encephalitogenic CD4+ T cells was antigen specific, their migration did not seem to be directional and was more like the random motility of naive T cells found in the lymph node (25).
In contrast to EAE induced by the transfer of autoimmune T cells and intracerebral injection of LCMV, a condition with well-defined localized acute inflammatory events, mice infected with T. gondii have provided a model of chronic CNS inflammation to study the behavior of pathogen-specific CD8+ T cells (28, 137). Unlike the rapid burst of infiltration during EAE, in this experimental system, there was a continuous recruitment of antigen-specific T cells that could be observed over a prolonged period of time (1–8 weeks) (28, 137). Various migratory behaviors including clustering and homotypic T cell interactions were observed. Slowing of CD8+ T cells and clustering of antigen-specific cells were seen around actively replicating parasites but not latent cysts (137). With no correlation between the amount of antigen present and the confinement ratio (or meandering index) of cells, the pattern of motility seemed to represent a “search and destroy mission” to find infected cells, rather than directional migration in response to a chemokine gradient.
Despite extensive investigations, we still have a limited understanding of exactly how chemokines and other chemotactic factors contribute to the migratory behavior of T cells, whether in meninges, CSF, or parenchyma. Since the initial studies describing the random behavior of T cell migration in the lymph node, it has been established that cells follow chemokine-coated conduits and thus remain “directed” (138). Previously, intravital imaging of peripheral lymph nodes indicated that naive T cells migrate at speeds greater than 10 μm/min and are guided by conduits formed by follicular dendritic cells, fibroblastic reticular cells, and stromal cells expressing the fibroblast marker ERTR7 (138). ERTR7+ cells have also been detected at distinct but confined areas of the brain (such as the meninges, vasculature, and sulci) during inflammation caused by LCMV (64) and T. gondii infection (28). There is little understanding of the role that ERTR7+ stromal cells have in the CNS, but it is tempting to speculate that these cells promote trafficking or retain migratory leukocytes in these distinct compartments of the brain.
While the presence of a haptotaxic mechanism of migration (i.e., migration in response to chemokine bound to matrix molecules) has given rise to the idea of random exploration, it has not excluded the role of soluble chemokine gradients, nor has it been shown that gradients exist in an immobilized fashion on stromal networks (139). However, this has led to investigations into the existence of similar networks in non-lymphoid organs. Indeed, the ECM in the CNS may have a similar role to that of the constitutive structures in the lymph node (28, 138). Inflammation in the brain and in the periphery induces the production of ECM molecules that are known to support cell migration in the context of neural development (140, 141). A proteomics study demonstrated the production of many ECM molecules by astrocytes (142), and increased expression of collagen and laminins associated with myelin-containing macrophages is present in perivascular lesions of patients with MS (143). The use of second harmonic generation signals during multi-photon microscopy led to the visualization of a reticular network of fibers in the inflamed brain that closely associated with migrating T cells (28). These fibers were not present in the brains or spinal cord of naive mice but were upregulated during T. gondii infection and following EAE induction. This network may be the functional equivalent of the fibroblastic reticular cell network in the lymph node (138) and might not only provide structural support for migration but also display bound chemotactic signals for directional migration of lymphocytes. This model needs to be rigorously tested, but it may explain how lymphocytes can reach migratory velocities in this dense tissue that are comparable to those of naive T cells within lymph nodes.